Intron sequence analysis method for detection of adjacent and remote locus alleles as haplotypes

ABSTRACT

The present invention provides a method for detection of at least one allele of a genetic locus and can be used to provide direct determination of the haplotype. The method comprises amplifying genomic DNA with a primer pair that spans an intron sequence and defines a DNA sequence in genetic linkage with an allele to be detected. The primer-defined DNA sequence contains a sufficient number of intron sequence nucleotides to characterize the allele. Genomic DNA is amplified to produce an amplified DNA sequence characteristic of the allele. The amplified DNA sequence is analyzed to detect the presence of a genetic variation in the amplified DNA sequence such as a change in the length of the sequence, gain or loss of a restriction site or substitution of a nucleotide. The variation is characteristic of the allele to be detected and can be used to detect remote alleles. Kits comprising one or more of the reagents used in the method are also described.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part of U.S. application Ser. No.07/465,863, filed Jan. 16, 1990, abandoned, which application is acontinuation-in-part of U.S. application Ser. No. 07/405,499, filed Sep.11, 1989, abandoned, which application is a continuation-in-part of U.S.application Ser. No. 07/398,217 filed Aug. 25, 1989, abandoned, whichapplications are entitled IMPROVED HLA TYPING METHOD AND REAGENTSTHEREFOR by Malcolm J. Simons. Each of those applications isincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to a method for detection of alleles andhaplotypes and reagents therefor.

BACKGROUND OF THE INVENTION

Due in part to a number of new analytical techniques, there has been asignificant increase in knowledge about genetic information,particularly in humans. Allelic variants of genetic loci have beencorrelated to malignant and non-malignant monogenic and multigenicdiseases. For example, monogenic diseases for which the defective genehas been identified include DuChenne muscular dystrophy, sickle-cellanemia, Lesch Nyhan syndrome, hemophilia, beta-thalassemia, cysticfibrosis, polycystic kidney disease, ADA deficiency, α-1-antitrypsindeficiency, Wilm's tumor and retinoblastoma. Other diseases which arebelieved to be monogenic for which the gene has not been identifiedinclude fragile X mental retardation and Huntington's chorea.

Genes associated with multigenic diseases such as diabetes, colon cancerand premature coronary atherosclerosis have also been identified.

In addition to identifying individuals at risk for or carriers ofgenetic diseases, detection of allelic variants of a genetic locus hasbeen used for organ transplantation, forensics, disputed paternity and avariety of other purposes in humans. In commercially important plantsand animals, genes have not only been analyzed but geneticallyengineered and transmitted into other organisms.

A number of techniques have been employed to detect allelic variants ofgenetic loci including analysis of restriction fragment lengthpolymorphic (RFLP) patterns, use of oligonucleotide probes, and DNAamplification methods. One of the most complicated groups of allelicvariants, the major histocompatibility complex (MHC), has beenextensively studied. The problems encountered in attempting to determinethe HLA type of an individual are exemplary of problems encountered incharacterizing other genetic loci.

The major histocompatibility complex is a cluster of genes that occupy aregion on the short arm of chromosome 6. This complex, denoted the humanleukocyte antigen (HLA) complex, includes at least 50 loci. For thepurposes of HLA tissue typing, two main classes of loci are recognized.The Class I loci encode transplantation antigens and are designated A, Band C. The Class II loci (DRA, DRB, DQA1, DQB, DPA and DPB) encodeproducts that control immune responsivenes. Of the Class II loci, allthe loci are polymorphic with the exception of the DRA locus. That is,the DRα antigen polypeptide sequence is invariant.

HLA determinations are used in paternity determinations, transplantcompatibility testing, forensics, blood component therapy,anthropological studies, and in disease association correlations todiagnose disease or predict disease susceptibility. Due power of HLA todistinguish individuals and the need to match HLA type fortransplantation, analytical methods to unambiguously characterize thealleles of the genetic loci associated with the complex have beensought. At present, DNA typing using RFLP and oligonucleotide probes hasbeen used to type Class II locus alleles. Alleles of Class I loci andClass II DR and DQ loci are typically determined by serological methods.The alleles of the Class II DP locus are determined by primed lymphocytetyping (PLT).

Each of the HLA analysis methods has drawbacks. Serological methodsrequire standard sera that are not widely available and must becontinuously replenished. Additionally, serotyping is based on thereaction of the HLA gene products in the sample with the antibodies inthe reagent sera. The antibodies recognize the expression products ofthe HLA genes on the surface of nucleated cells. The determination offetal HLA type by serological methods may be difficult due to lack ofmaturation of expression of the antigens in fetal blood cells.

Oligonucleotide probe typing can be performed in two days and has beenfurther improved by the recent use of polymerase chain reaction (PCR)amplification. PCR-based oligoprobe typing has been performed on ClassII loci. Primed lymphocyte typing requires 5 to 10 days to complete andinvolves cell culture with its difficulties and inherent variability.

RFLP analysis is time consuming, requiring about 5 to 7 days tocomplete. Analysis of the fragment patterns is complex. Additionally,the technique requires the use of labelled probes. The most commonlyused label, ³² P, presents well known drawbacks associated with the useof radionuclides.

A fast, reliable method of genetic locus analysis is highly desirable.

DESCRIPTION OF THE PRIOR ART

U.S. Pat. No. 4,683,195 (to Mullis et al, issued Jul. 28, 1987)describes a process for amplifying, detecting and/or cloning nucleicacid sequences. The method involves treating separate complementarystrands of DNA with two oligonucleotide primers, extending the primersto form complementary extension products that act as templates forsynthesizing the desired nucleic acid sequence and detecting theamplified sequence. The method is commonly referred to as the polymerasechain reaction sequence amplification method or PCR. Variations of themethod are described in U.S. Pat. No. 4,683,194 (to Saiki et al, issuedJul. 28, 1987). The polymerase chain reaction sequence amplificationmethod is also described by Saiki et al, Science, 230:1350-1354 (1985)and Scharf et al, Science, 324:163-166 (1986).

U.S. Pat. No. 4,582,788 (to Erlich, issued Apr. 15, 1986) describes anHLA typing method based on restriction length polymorphism (RFLP) andcDNA probes used therewith. The method is carried out by digesting anindividual's HLA DNA with a restriction endonuclease that produces apolymorphic digestion pattern, subjecting the digest to genomic blottingusing a labelled cDNA probe that is complementary to an HLA DNA sequenceinvolved in the polymorphism, and comparing the resulting genomicblotting pattern with a standard. Locus-specific probes for Class IIloci (DQ) are also described.

Kogan et al, New Engl. J. Med, 317:985-990 (1987) describes an improvedPCR sequence amplification method that uses a heat-stable polymerase(Taq polymerase) and high temperature amplification. The stringentconditions used in the method provide sufficient fidelity of replicationto permit analysis of the amplified DNA by determining DNA sequencelengths by visual inspection of an ethidium bromide-stained gel. Themethod was used to analyze DNA associated with hemophilia A in whichadditional tandem repeats of a DNA sequence are associated with thedisease and the amplified sequences were significantly longer thansequences that are not associated with the disease.

Simons and Erlich, pp 952-958 In: Immunology of HLA Vol. 1:Springer-Verlag, New York (1989) summarized RFLP-sequence interrelationsat the DPA and DPB loci. RFLP fragment patterns analyzed with probes bySouthern blotting provided distinctive patterns for DPw1-5 alleles andthe corresponding DPB1 allele sequences, characterized two subtypicpatterns for DPw2 and DPw4, and identified new DPw alleles.

Simons et al, pp 959-1023 In: Immunology of HLA Vol. 1: Springer-Verlag,New York (1989) summarized restriction length polymorphisms of HLAsequences for class II loci as determined by the 10th InternationalWorkshop Southern Blot Analysis. Southern blot analysis was shown to besuitable for typing of the major classes of HLA loci.

A series of three articles [Rommens et al, Science 245:1059-1065 (1989),Riordan et al, Science 245:1066-1072 (1989) and Kerem et al, Science245:1073-1079 (1989) report a new gene analysis method called "jumping"used to identify the location of the CF gene, the sequence of the CFgene, and the defect in the gene and its percentage in the diseasepopulation, respectively.

DiLelia et al, The Lancet i:497-499 (1988) describes a screening methodfor detecting the two major alleles responsible for phenylketonuria incaucasians of Northern European descent. The mutations, located at aboutthe center of exon 12 and at the exon 12 junction with interveningsequence 12 are detected by PCR amplification of a 245 bp region of exon12 and flanking intervening sequences. The amplified sequenceencompasses both mutations and is analyzed using probes specific foreach of the alleles (without prior electrophoretic separation).

Dicker et al, BioTechniques 7:830-837 (1989) and Mardis et al,BioTechniques 7:840-850 (1989) report on automated techniques forsequencing of DNA sequences, particularly PCR-generated sequences.

Each of the above-described references is incorporated herein byreference in its entirety.

SUMMARY OF THE INVENTION

The present invention provides a method for detection of at least oneallele of a genetic locus and can be used to provide directdetermination of the haplotype. The method comprises amplifying genomicDNA with a primer pair that spans an intron sequence and defines a DNAsequence in genetic linkage with an allele to be detected. Theprimer-defined DNA sequence contains a sufficient number of intronsequence nucleotides to characterize the allele. Genomic DNA isamplified to produce an amplified DNA sequence characteristic of theallele. The amplified DNA sequence is analyzed to detect the presence ofa genetic variation in the amplified DNA sequence such as a change inthe length of the sequence, gain or loss of a restriction site orsubstitution of a nucleotide. The variation is characteristic of theallele to be detected.

The present invention is based on the finding that intron sequencescontain genetic variations that are characteristic of adjacent andremote alleles on the same chromosome. In particular, DNA sequences thatinclude a sufficient number of intron sequence nucleotides can be usedfor direct determination of haplotype.

The method can be used to detect alleles of genetic loci for anyeukaryotic organism. Of particular interest are loci associated withmalignant and nonmalignant monogenic and multigenic diseases, andidentification of individual organisms or species in both plants andanimals. In a preferred embodiment, the method is used to determine HLAallele type and haplotype.

Kits comprising one or more of the reagents used in the method are alsodescribed.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method for detection of alleles andhaplotypes through analysis of intron sequence variation. The presentinvention is based on the discovery that amplification of intronsequences that exhibit linkage disequilibrium with adjacent and remoteloci can be used to detect alleles of those loci. The present methodreads haplotypes as the direct output of the intron typing analysis whena single, individual organism is tested. The method is particularlyuseful in humans but is generally applicable to all eukaryotes, and ispreferably used to analyze plant and animal species.

The method comprises amplifying genomic DNA with a primer pair thatspans an intron sequence and defines a DNA sequence in genetic linkagewith an allele to be detected. Primer sites are located in conservedregions in the introns or exons bordering the intron sequence to beamplified. The primer-defined DNA sequence contains a sufficient numberof intron sequence nucleotides to characterize the allele. The amplifiedDNA sequence is analyzed to detect the presence of a genetic variationsuch as a change in the length of the sequence, gain or loss of arestriction site or substitution of a nucleotide.

The intron sequences provide genetic variations that, in addition tothose found in exon sequences, further distinguish sample DNA, providingadditional information about the individual organism. This informationis particularly valuable for identification of individuals such as inpaternity determinations and in forensic applications. The informationis also valuable in any other application where heterozygotes (twodifferent alleles) are to be distinguished from homozygotes (two copiesof one allele).

More specifically, the present invention provides information regardingintron variation. Using the methods and reagents of this invention, twotypes of intron variation associated with genetic loci have been found.The first is allele-associated intron variation. That is, the intronvariation pattern associates with the allele type at an adjacent locus.The second type of variation is associated with remote alleles(haplotypes). That is, the variation is present in individual organismswith the same genotype at the primary locus. Differences may occurbetween sequences of the same adjacent and remote locus types. However,individual-limited variation is uncommon.

Furthermore, an amplified DNA sequence that contains sufficient intronsequences will vary depending on the allele present in the sample. Thatis, the introns contain genetic variations (e.g. length polymorphismsdue to insertions and/or deletions and changes in the number or locationof restriction sites) which are associated with the particular allele ofthe locus and with the alleles at remote loci.

The reagents used in carrying out the methods of this invention are alsodescribed. The reagents can be provided in kit form comprising one ormore of the reagents used in the method.

Definitions

The term "allele", as used herein, means a genetic variation associatedwith a coding region; that is, an alternative form of the gene.

The term "linkage", as used herein, refers to the degree to whichregions of genomic DNA are inherited together. Regions on differentchromosomes do not exhibit linkage and are inherited together 50% of thetime. Adjacent genes that are always inherited together exhibit 100%linkage.

The term "linkage disequilibrium", as used herein, refers to theco-occurrence of two alleles at linked loci such that the frequency ofthe co-occurrence of the alleles is greater than would be expected fromthe separate frequencies of occurrence of each allele. Alleles thatco-occur with frequencies expected from their separate frequencies aresaid to be in "linkage equilibrium".

As used herein, "haplotype" is a region of genomic DNA on a chromosomewhich is bounded by recombination sites such that genetic loci within ahaplotypic region are usually inherited as a unit. However,occasionally, genetic rearrangements may occur within a haplotype. Thus,the term haplotype is an operational term that refers to the occurrenceon a chromosome of linked loci.

As used herein, the term "intron" refers to untranslated DNA sequencesbetween exons, together with 5' and 3' untranslated regions associatedwith a genetic locus. In addition, the term is used to refer to thespacing sequences between genetic loci (intergenic spacing sequences)which are not associated with a coding region and are colloquiallyreferred to as "junk". While the art traditionally uses the term"intron" to refer only to untranslated sequences between exons, thisexpanded definition was necessitated by the lack of any art recognizedterm which encompasses all non-exon sequences.

As used herein, an "intervening sequence" is an intron which is locatedbetween two exons within a gene. The term does not encompass upstreamand downstream noncoding sequences associated with the genetic locus.

As used herein, the term "amplified DNA sequence" refers to DNAsequences which are copies of a portion of a DNA sequence and itscomplementary sequence, which copies correspond in nucleotide sequenceto the original DNA sequence and its complementary sequence.

The term "complement", as used herein, refers to a DNA sequence that iscomplementary to a specified DNA sequence.

The term "primer site", as used herein, refers to the area of the targetDNA to which a primer hybridizes.

The term "primer pair", as used herein, means a set of primers includinga 5' upstream primer that hybridizes with the 5' end of the DNA sequenceto be amplified and a 3' downstream primer that hybridizes with thecomplement of the 3' end of the sequence to be amplified.

The term "exon-limited primers", as used herein, means a primer pairhaving primers located within or just outside of an exon in a conservedportion of the intron, which primers amplify a DNA sequence whichincludes an exon or a portion thereof and not more than a small,para-exon region of the adjacent intron(s).

The term "intron-spanning primers", as used herein, means a primer pairthat amplifies at least a portion of one intron, which amplified intronregion includes sequences which are not conserved. The intron-spanningprimers can be located in conserved regions of the introns or inadjacent, upstream and/or downstream exon sequences.

The term "genetic locus", as used herein, means the region of thegenomic DNA that includes the gene that encodes a protein including anyupstream or downstream transcribed noncoding regions and associatedregulatory regions. Therefore, an HLA locus is the region of the genomicDNA that includes the gene that encodes an HLA gene product.

As used herein, the term "adjacent locus" refers to either (1) the locusin which a DNA sequence is located or (2) the nearest upstream ordownstream genetic locus for intron DNA sequences not associated with agenetic locus.

As used herein, the term "remote locus" refers to either (1) a locuswhich is upstream or downstream from the locus in which a DNA sequenceis located or (2) for intron sequences not associated with a geneticlocus, a locus which is upstream or downstream from the nearest upstreamor downstream genetic locus to the intron sequence.

The term "locus-specific primer", as used herein, means a primer thatspecifically hybridizes with a portion of the stated gene locus or itscomplementary strand, at least for one allele of the locus, and does nothybridize with other DNA sequences under the conditions used in theamplification method.

As used herein, the terms "endonuclease" and "restriction endonuclease"refer to an enzyme that cuts double-stranded DNA having a particularnucleotide sequence. The specificities of numerous endonucleases arewell known and can be found in a variety of publications, e.g. MolecularCloning: A Laboratory Manual by Maniatis et al, Cold Spring HarborLaboratory 1982. That manual is incorporated herein by reference in itsentirety.

The term "restriction fragment length polymorphism" (or RFLP), as usedherein, refers to differences in DNA nucleotide sequences that producefragments of different lengths when cleaved by a restrictionendonuclease.

The term "primer-defined length polymorphisms" (or PDLP), as usedherein, refers to differences in the lengths of amplified DNA sequencesdue to insertions or deletions in the intron region of the locusincluded in the amplified DNA sequence.

The term "HLA DNA", as used herein, means DNA that includes the genesthat encode HLA antigens. HLA DNA is found in all nucleated human cells.

Primers

The method of this invention is based on amplification of selectedintron regions of genomic DNA. The methodology is facilitated by the useof primers that selectively amplify DNA associated with one or morealleles of a genetic locus of interest and not with other genetic loci.

A locus-specific primer pair contains a 5' upstream primer that definesthe 5' end of the amplified sequence by hybridizing with the 5' end ofthe target sequence to be amplified and a 3' downstream primer thatdefines the 3' end of the amplified sequence by hybridizing with thecomplement of the 3' end of the DNA sequence to be amplified. Theprimers in the primer pair do not hybridize with DNA of other geneticloci under the conditions used in the present invention.

For each primer of the locus-specific primer pair, the primer hybridizesto at least one allele of the DNA locus to be amplified or to itscomplement. A primer pair can be prepared for each allele of a selectedlocus, which primer pair amplifies only DNA for the selected locus. Inthis way combinations of primer pairs can be used to amplify genomic DNAof a particular locus, irrespective of which allele is present in asample. Preferably, the primer pair amplifies DNA of at least two, morepreferably more than two, alleles of a locus. In a most preferredembodiment, the primer sites are conserved, and thus amplify allhaplotypes. However, primer pairs or combinations thereof thatspecifically bind with the most common alleles present in a particularpopulation group are also contemplated.

The amplified DNA sequence that is defined by the primers contains asufficient number of intron sequence nucleotides to distinguish betweenat least two alleles of an adjacent locus, and preferably, to identifythe allele of the locus which is present in the sample. For somepurposes, the sequence can also be selected to contain sufficientgenetic variations to distinguish between individual organisms with thesame allele or to distinguish between haplotypes.

Length of sequence

The length of the amplified sequence which is required to includesufficient genetic variability to enable discrimination between allalleles of a locus bears a direct relation to the extent of thepolymorphism of the locus (the number of alleles). That is, as thenumber of alleles of the tested locus increases, the size of anamplified sequence which contains sufficient genetic variations toidentify each allele increases. For a particular population group, oneor more of the recognized alleles for any given locus may be absent fromthat group and need not be considered in determining a sequence whichincludes sufficient variability for that group. Conveniently, however,the primer pairs are selected to amplify a DNA sequence which issufficient to distinguish between all recognized alleles of the testedlocus. The same considerations apply when a haplotype is determined.

For example, the least polymorphic HLA locus is DPA which currently hasfour recognized alleles. For that locus, a primer pair which amplifiesonly a portion of the variable exon encoding the allelic variationcontains sufficient genetic variability to distinguish between thealleles when the primer sites are located in an appropriate region ofthe variable exon. Exonlimited primers can be used to produce anamplified sequence that includes as few as about 200 nucleotides (nt).However, as the number of alleles of the locus increases, the number ofgenetic variations in the sequence must increase to distinguish allalleles. Addition of invariant exon sequences provides no additionalgenetic variation. When about eight or more alleles are to bedistinguished, as for the DQA1 locus and more variable loci, amplifiedsequences should extend into at least one intron in the locus,preferably an intron adjacent to the variable exon.

Additionally, where alleles of the locus exist which differ by a singlebasepair in the variable exon, intron sequences are included inamplified sequences to provide sufficient variability to distinguishalleles. For example, for the DQA1 locus (with eight currentlyrecognized alleles) and the DPB locus (with 24 alleles), the DQA1.1/1.2(now referred to as DQA1 0101/0102) and DPB2.1/4.2 (now referred to asDPB0201/0402) alleles differ by a single basepair. To distinguish thosealleles, amplified sequences which include an intron sequence region arerequired. About 300 to 500 nucleotides is sufficient, depending on thelocation of the sequence. That is, 300 to 500 nucleotides comprisedprimarily of intron sequence nucleotides sufficiently close to thevariable exon are sufficient.

For loci with more extensive polymorphisms (such as DQB with 14currently recognized alleles, DPB with 24 currently recognized alleles,DRB with 34 currently recognized alleles and for each of the Class Iloci), the amplified sequences need to be larger to provide sufficientvariability to distinguish between all the alleles. An amplifiedsequence that includes at least about 0.5 kilobases (Kb), preferably atleast about 1.0 Kb, more preferably at least about 1.5 Kb generallyprovides a sufficient number of restriction sites for loci withextensive polymorphisms. The amplified sequences used to characterizehighly polymorphic loci are generally between about 800 to about 2,000nucleotides (nt), preferably between about 1000 to about 1800nucleotides in length.

When haplotype information regarding remote alleles is desired, thesequences are generally between about 1,000 to about 2,000 nt in length.Longer sequences are required when the amplified sequence encompasseshighly conserved regions such as exons or highly conserved intronregions, e.g., promoters, operators and other DNA regulatory regions.Longer amplified sequences (including more intron nucleotide sequences)are also required as the distance between the amplified sequences andthe allele to be detected increases.

Highly conserved regions included in the amplified DNA sequence, such asexon sequences or highly conserved intron sequences (e.g. promoters,enhancers, or other regulatory regions) may provide little or no geneticvariation. Therefore, such regions do not contribute, or contribute onlyminimally, to the genetic variations present in the amplified DNAsequence. When such regions are included in the amplified DNA sequence,additional nucleotides may be required to encompass sufficient geneticvariations to distinguish alleles, in comparison to an amplified DNAsequence of the same length including only intron sequences.

Location of the amplified DNA sequence

The amplified DNA sequence is located in a region of genomic DNA thatcontains genetic variation which is in genetic linkage with the alleleto be detected. Preferably, the sequence is located in an intronsequence adjacent to an exon of the genetic locus. More preferably, theamplified sequence includes an intervening sequence adjacent to an exonthat encodes the allelic variability associated with the locus (avariable exon). The sequence preferably includes at least a portion ofone of the introns adjacent to a variable exon and can include a portionof the variable exon. When additional sequence information is required,the amplified DNA sequence preferably encompasses a variable exon andall or a portion of both adjacent intron sequences.

Alternatively, the amplified sequence can be in an intron which does notborder an exon of the genetic locus. Such introns are located in thedownstream or upstream gene flanking regions or even in an interveningsequence in another genetic locus which is in linkage disequilibriumwith the allele to be detected.

For some genetic loci, genomic DNA sequences may not be available. Whenonly cDNA sequences are available and intron locations within thesequence are not identified, primers are selected at intervals of about200 nt and used to amplify genomic DNA. If the amplified sequencecontains about 200 nt, the location of the first primer is moved about200 nt to one side of the second primer location and the amplificationis repeated until either (1) an amplified DNA sequence that is largerthan expected is produced or (2) no amplified DNA sequence is produced.In either case, the location of an intron sequence has been determined.The same methodology can be used when only the sequence of a marker sitethat is highly linked to the genetic locus is available, as is the casefor many genes associated with inherited diseases.

When the amplified DNA sequence does not include all or a portion of anintron adjacent to the variable exon(s), the sequence must also satisfya second requirement. The amplified sequence must be sufficiently closeto the variable exon(s) to exclude recombination and loss of linkagedisequilibrium between the amplified sequence and the variable exon(s).This requirement is satisfied if the regions of the genomic DNA arewithin about 5 Kb, preferably within about 4 Kb, most preferably within2 Kb of the variable exon(s). The amplified sequence can be outside ofthe genetic locus but is preferably within the genetic locus.

Preferably, for each primer pair, the amplified DNA sequence defined bythe primers includes at least 200 nucleotides, and more preferably atleast 400 nucleotides, of an intervening sequence adjacent to thevariable exon(s). Although the variable exon usually provides fewervariations in a given number of nucleotides than an adjacent interveningsequence, each of those variations provides allele-relevant information.Therefore, inclusion of the variable exon provides an advantage.

Since PCR methodology can be used to amplify sequences of several Kb,the primers can be located so that additional exons or interveningsequences are included in the amplified sequence. Of course, theincreased size of the amplified DNA sequence increases the chance ofreplication error, so addition of invariant regions provides somedisadvantages. However, those disadvantages are not as likely to affectan analysis based on the length of the sequence or the RFLP fragmentpatterns as one based on sequencing the amplification product. Forparticular alleles, especially those with highly similar exon sequences,amplified sequences of greater than about 1 or 1.5 Kb may be necessaryto discriminate between all alleles of a particular locus.

The ends of the amplified DNA sequence are defined by the primer pairused in the amplification. Each primer sequence must correspond to aconserved region of the genomic DNA sequence. Therefore, the location ofthe amplified sequence will, to some extent, be dictated by the need tolocate the primers in conserved regions. When sufficient intron sequenceinformation to determine conserved intron regions is not available, theprimers can be located in conserved portions of the exons and used toamplify intron sequences between those exons.

When appropriately-located, conserved sequences are not unique to thegenetic locus, a second primer located within the amplified sequenceproduced by the first primer pair can be used to provide an amplifiedDNA sequence specific for the genetic locus. At least one of the primersof the second primer pair is located in a conserved region of theamplified DNA sequence defined by the first primer pair. The secondprimer pair is used following amplification with the first primer pairto amplify a portion of the amplified DNA sequence produced by the firstprimer pair.

There are three major types of genetic variations that can be detectedand used to identify an allele. Those variations, in order of ease ofdetection, are (1) a change in the length of the sequence, (2) a changein the presence or location of at least one restriction site and (3) thesubstitution of one or a few nucleotides that does not result in achange in a restriction site. Other variations within the amplified DNAsequence are also detectable.

There are three types of techniques which can be used to detect thevariations. The first is sequencing the amplified DNA sequence.Sequencing is the most time consuming and also the most revealinganalytical method, since it detects any type of genetic variation in theamplified sequence. The second analytical method uses allele-specificoligonucleotide or sequence-specific oligonucleotides probes (ASO or SSOprobes). Probes can detect single nucleotide changes which result in anyof the types of genetic variations, so long as the exact sequence of thevariable site is known. A third type of analytical method detectssequences of different lengths (e.g., due to an insertion or deletion ora change in the location of a restriction site) and/or different numbersof sequences (due to either gain or loss of restriction sites). Apreferred detection method is by gel or capillary electrophoresis. Todetect changes in the lengths of fragments or the number of fragmentsdue to changes in restriction sites, the amplified sequence must bedigested with an appropriate restriction endonuclease prior to analysisof fragment length patterns.

The first genetic variation is a difference in the length of theprimer-defined amplified DNA sequence, referred to herein as aprimer-defined length polymorphism (PDLP), which difference in lengthdistinguishes between at least two alleles of the genetic locus. ThePDLPs result from insertions or deletions of large stretches (incomparison to the total length of the amplified DNA sequence) of DNA inthe portion of the intron sequence defined by the primer pair. To detectPDLPs, the amplified DNA sequence is located in a region containinginsertions or deletions of a size that is detectable by the chosenmethod. The amplified DNA sequence should have a length which providesoptimal resolution of length differences. For electrophoresis, DNAsequences of about 300 to 500 bases in length provide optimal resolutionof length differences. Nucleotide sequences which differ in length by asfew as 3 nt, preferably 25 to 50 nt, can be distinguished. However,sequences as long as 800 to 2,000 nt which differ by at least about 50nt are also readily distinguishable. Gel electrophoresis and capillaryelectrophoresis have similar limits of resolution. Preferably the lengthdifferences between amplified DNA sequences will be at least 10, morepreferably 20, most preferably 50 or more, nt between the alleles.Preferably, the amplified DNA sequence is between 300 to 1,000 nt andencompasses length differences of at least 3, preferably 10 or more nt.

Preferably, the amplified sequence is located in an area which providesPDLP sequences that distinguish most or all of the alleles of a locus.An example of PDLP-based identification of five of the eight DQA1alleles is described in detail in the examples.

When the variation to be detected is a change in a restriction site, theamplified DNA sequence necessarily contains at least one restrictionsite which (1) is present in one allele and not in another, (2) isapparently located in a different position in the sequence of at leasttwo alleles, or (3) combinations thereof. The amplified sequence willpreferably be located such that restriction endonuclease cleavageproduces fragments of detectably different lengths, rather than two ormore fragments of approximately the same length.

For allelic differences detected by ASO or SSO probes, the amplified DNAsequence includes a region of from about 200 to about 400 nt which ispresent in one or more alleles and not present in one or more otheralleles. In a most preferred embodiment, the sequence contains a regiondetectable by a probe that is present in only one allele of the geneticlocus. However, combinations of probes which react with some alleles andnot others can be used to characterize the alleles.

For the method described herein, it is contemplated that use of morethan one amplified DNA sequence and/or use of more than one analyticalmethod per amplified DNA sequence may be required for highly polymorphicloci, particularly for loci where alleles differ by single nucleotidesubstitutions that are not unique to the allele or when informationregarding remote alleles (haplotypes) is desired. More particularly, itmay be necessary to combine a PDLP analysis with an RFLP analysis, touse two or more amplified DNA sequences located in different positionsor to digest a single amplified DNA sequence with a plurality ofendonucleases to distinguish all the alleles of some loci. Thesecombinations are intended to be included within the scope of thisinvention.

For example, the analysis of the haplotypes of DQA1 locus described inthe examples uses PDLPs and RFLP analysis using three different enzymedigests to distinguish the eight alleles and 20 of the 32 haplotypes ofthe locus.

Length and sequence homology of primers

Each locus-specific primer includes a number of nucleotides which, underthe conditions used in the hybridization, are sufficient to hybridizewith an allele of the locus to be amplified and to be free fromhybridization with alleles of other loci. The specificity of the primerincreases with the number of nucleotides in its sequence underconditions that provide the same stringency. Therefore, longer primersare desirable. Sequences with fewer than 15 nucleotides are less certainto be specific for a particular locus. That is, sequences with fewerthan 15 nucleotides are more likely to be present in a portion of theDNA associated with other genetic loci, particularly loci of othercommon origin or evolutionarily closely related origin, in inverseproportion to the length of the nucleotide sequence.

Each primer preferably includes at least about 15 nucleotides, morepreferably at least about 20 nucleotides. The primer preferably does notexceed about 30 nucleotides, more preferably about 25 nucleotides. Mostpreferably, the primers have between about 20 and about 25 nucleotides.

A number of preferred primers are described herein. Each of thoseprimers hybridizes with at least about 15 consecutive nucleotides of thedesignated region of the allele sequence. For many of the primers, thesequence is not identical for all of the other alleles of the locus. Foreach of the primers, additional preferred primers have sequences whichcorrespond to the sequences of the homologous region of other alleles ofthe locus or to their complements.

When two sets of primer pairs are used sequentially, with the secondprimer pair amplifying the product of the first primer pair, the primerscan be the same size as those used for the first amplification. However,smaller primers can be used in the second amplification and provide therequisite specificity. These smaller primers can be selected to beallele-specific, if desired. The primers of the second primer pair canhave 15 or fewer, preferably 8 to 12, more preferably 8 to 10nucleotides. When two sets of primer pairs are used to produce twoamplified sequences, the second amplified DNA sequence is used in thesubsequent analysis of genetic variation and must meet the requirementsdiscussed previously for the amplified DNA sequence.

The primers preferably have a nucleotide sequence that is identical to aportion of the DNA sequence to be amplified or its complement. However,a primer having two nucleotides that differ from the target DNA sequenceor its complement also can be used. Any nucleotides that are notidentical to the sequence or its complement are preferably not locatedat the 3' end of the primer. The 3' end of the primer preferably has atleast two, preferably three or more, nucleotides that are complementaryto the sequence to which the primer binds. Any nucleotides that are notidentical to the sequence to be amplified or its complement willpreferably not be adjacent in the primer sequence. More preferably,noncomplementary nucleotides in the primer sequence will be separated byat least three, more preferably at least five, nucleotides. The primersshould have a melting temperature (T_(m)) from about 55° to 75° C.Preferably the T_(m) is from about 60° C. to about 65° C. to facilitatestringent amplification conditions.

The primers can be prepared using a number of methods, such as, forexample, the phosphotriester and phosphodiester methods or automatedembodiments thereof. The phosphodiester and phosphotriester methods aredescribed in Cruthers, Science 230:281-285 (1985); Brown et al. Meth.Enzymol., 68:109 (1979); and Nrang et al, Meth. Enzymol., 68:90 (1979).In one automated method, diethylphosphoramidites which can besynthesized as described by Beaucage et al, Tetrahedron letters,22:1859-1962 (1981) are used as starting materials. A method forsynthesizing primer oligonucleotide sequences on a modified solidsupport is described in U.S. Pat. No. 4,458,066. Each of the abovereferences is incorporated herein by reference in its entirety.

Exemplary primer sequences for analysis of Class I and Class II HLAloci; bovine leukocyte antigens, and cystic fibrosis are describedherein.

Amplification

The locus-specific primers are used in an amplification process toproduce a sufficient amount of DNA for the analysis method. Forproduction of RFLP fragment patterns or PDLP patterns which are analyzedby electrophoresis, about 1 to about 500 Ng of DNA is required. Apreferred amplification method is the polymerase chain reaction (PCR).PCR amplification methods are described in U.S. Pat. No. 4,683,195 (toMullis et al, issued Jul. 28, 1987); U.S. Pat. No. 4,683,194 (to Saikiet al, issued Jul. 28, 1987); Saiki et al. Science, 230:1350-1354(1985); Scharf et al, Science, 324:163-166 (1986); Kogan et al, NewEngl. J. Med, 317:985-990 (1987) and Saiki, Gyllensten and Erlich, ThePolymerase Chain Reaction in Genome Analysis: A Practical Approach, ed.Davies pp. 141-152, (1988) I.R.L. Press, Oxford. Each of the abovereferences is incorporated herein by reference in its entirety.

Prior to amplification, a sample of the individual organism's DNA isobtained. All nucleated cells contain genomic DNA and, therefore, arepotential sources of the required DNA. For higher animals, peripheralblood cells are typically used rather than tissue samples. As little as0.01 to 0.05 cc of peripheral blood provides sufficient DNA foramplification. Hair, semen and tissue can also be used as samples. Inthe case of fetal analyses, placental cells or fetal cells present inamniotic fluid can be used. The DNA is isolated from nucleated cellsunder conditions that minimize DNA degradation. Typically, the isolationinvolves digesting the cells with a protease that does not attack DNA ata temperature and pH that reduces the likelihood of DNase activity. Forperipheral blood cells, lysing the cells with a hypotonic solution(water) is sufficient to release the DNA.

DNA isolation from nucleated cells is described by Kan et al, N. Engl.J. Med. 297:1080-1084 (1977); Kan et al, Nature 251:392-392 (1974); andKan et al, PNAS 75:5631-5635 (1978). Each of the above references isincorporated herein by reference in its entirety. Extraction proceduresfor samples such as blood, semen, hair follicles, semen, mucous membraneepithelium and other sources of genomic DNA are well known. For plantcells, digestion of the cells with cellulase releases DNA. ThereafterDNA is purified as described above.

The extracted DNA can be purified by dialysis, chromatography, or otherknown methods for purifying polynucleotides prior to amplification.Typically, the DNA is not purified prior to amplification.

The amplified DNA sequence is produced by using the portion of the DNAand its complement bounded by the primer pair as a template. As a firststep in the method, the DNA strands are separated into single strandedDNA. This strand separation can be accomplished by a number of methodsincluding physical or chemical means. A preferred method is the physicalmethod of separating the strands by heating the DNA until it issubstantially (approximately 93%) denatured. Heat denaturation involvestemperatures ranging from about 80° to 105° C. for times ranging fromabout 15 to 30 seconds. Typically, heating the DNA to a temperature offrom 90° to 93° C. for about 30 seconds to about 1 minute is sufficient.

The primer extension product(s) produced are complementary to theprimer-defined region of the DNA and hybridize therewith to form aduplex of equal length strands. The duplexes of the extension productsand their templates are then separated into single-stranded DNA. Whenthe complementary strands of the duplexes are separated, the strands areready to be used as a template for the next cycle of synthesis ofadditional DNA strands.

Each of the synthesis steps can be performed using conditions suitablefor DNA amplification. Generally, the amplification step is performed ina buffered aqueous solution, preferably at a pH of about 7 to about 9,more preferably about pH 8. A suitable amplification buffer containsTris-HCl as a buffering agent in the range of about 10 to 100 mM. Thebuffer also includes a monovalent salt, preferably at a concentration ofat least about 10 mM and not greater than about 60 mM. Preferredmonovalent salts are KCl, NaCl and (NH₄)₂ SO₄. The buffer also containsMgCl₂ at about 5 to 50 mM. Other buffering systems such as hepes orglycine-NaOH and potassium phosphate buffers can be used. Typically, thetotal volume of the amplification reaction mixture is about 50 to 100μl.

Preferably, for genomic DNA, a molar excess of about 10⁶ :1primer:template of the primer pair is added to the buffer containing theseparated DNA template strands. A large molar excess of the primersimproves the efficiency of the amplification process. In general, about100 to 150 ng of each primer is added.

The deoxyribonucleotide triphosphates dATP, dCTP, dGTP and dTTP are alsoadded to the amplification mixture in amounts sufficient to produce theamplified DNA sequences. Preferably, the dNTPs are present at aconcentration of about 0.75 to about 4.0 mM, more preferably about 2.0mM. The resulting solution is heated to about 90° to 93° C. for fromabout 30 seconds to about 1 minute to separate the strands of the DNA.After this heating period the solution is cooled to the amplificationtemperature.

Following separation of the DNA strands, the primers are allowed toanneal to the strands. The annealing temperature varies with the lengthand GC content of the primers. Those variables are reflected in theT_(m) of each primer. Exemplary HLA DQA1 primers of this invention,described below, require temperatures of about 55° C. The exemplary HLAClass I primers of this invention require slightly higher temperaturesof about 62° to about 68° C. The extension reaction step is performedfollowing annealing of the primers to the genomic DNA.

An appropriate agent for inducing or catalyzing the primer extensionreaction is added to the amplification mixture either before or afterthe strand separation (denaturation) step, depending on the stability ofthe agent under the denaturation conditions. The DNA synthesis reactionis allowed to occur under conditions which are well known in the art.This synthesis reaction (primer extension) can occur at from roomtemperature up to a temperature above which the polymerase no longerfunctions efficiently. Elevating the amplification temperature enhancesthe stringency of the reaction. As stated previously, stringentconditions are necessary to ensure that the amplified sequence and theDNA template sequence contain the same nucleotide sequence, sincesubstitution of nucleotides can alter the restriction sites or probebinding sites in the amplified sequence.

The inducing agent may be any compound or system which facilitatessynthesis of primer extension products, preferably enzymes. Suitableenzymes for this purpose include DNA polymerases (such as, for example,E. coli DNA polymerase I, Klenow fragment of E. coli DNA polymerase I,T4 DNA polymerase), reverse transcriptase, and other enzymes (includingheat-stable polymerases) which facilitate combination of the nucleotidesin the proper manner to form the primer extension products. Mostpreferred is Taq polymerase or other heat-stable polymerases whichfacilitate DNA synthesis at elevated temperatures (about 60° to 90° C.).Taq polymerase is described, e.g., by Chien et al, J. Bacteriol.,127:1550-1557 (1976). That article is incorporated herein by referencein its entirety. When the extension step is performed at about 72° C.,about 1 minute is required for every 1000 bases of target DNA to beamplified.

The synthesis of the amplified sequence is initiated at the 3' end ofeach primer and proceeds toward the 5' end of the template along thetemplate DNA strand, until synthesis terminates, producing DNA sequencesof different lengths. The newly synthesized strand and its complementarystrand form a double-stranded molecule which is used in the succeedingsteps of the process. In the next step, the strands of thedouble-stranded molecule are separated (denatured) as described above toprovide single-stranded molecules.

New DNA is synthesized on the single-stranded template molecules.Additional polymerase, nucleotides and primers can be added if necessaryfor the reaction to proceed under the conditions described above. Afterthis step, half of the extension product consists of the amplifiedsequence bounded by the two primers. The steps of strand separation andextension product synthesis can be repeated as many times as needed toproduce the desired quantity of the amplified DNA sequence. The amountof the amplified sequence produced accumulates exponentially. Typically,about 25 to 30 cycles are sufficient to produce a suitable amount of theamplified DNA sequence for analysis.

The amplification method can be performed in a step-wise fashion whereafter each step new reagents are added, or simultaneously, where allreagents are added at the initial step, or partially step-wise andpartially simultaneously, where fresh reagent is added after a givennumber of steps. The amplification reaction mixture can contain, inaddition to the sample genomic DNA, the four nucleotides, the primerpair in molar excess, and the inducing agent, e.g., Taq polymerase.

Each step of the process occurs sequentially notwithstanding the initialpresence of all the reagents. Additional materials may be added asnecessary. Typically, the polymerase is not replenished when using aheat-stable polymerase. After the appropriate number of cycles toproduce the desired amount of the amplified sequence, the reaction maybe halted by inactivating the enzymes, separating the components of thereaction or stopping the thermal cycling.

In a preferred embodiment of the method, the amplification includes theuse of a second primer pair to perform a second amplification followingthe first amplification. The second primer pair defines a DNA sequencewhich is a portion of the first amplified sequence. That is, at leastone of the primers of the second primer pair defines one end of thesecond amplified sequence which is within the ends of the firstamplified sequence. In this way, the use of the second primer pair helpsto ensure that any amplified sequence produced in the secondamplification reaction is specific for the tested locus. That is,non-target sequences which may be copied by a locus-specific pair areunlikely to contain sequences that hybridize with a secondlocus-specific primer pair located within the first amplified sequence.

In another embodiment, the second primer pair is specific for one alleleof the locus. In this way, detection of the presence of a secondamplified sequence indicates that the allele is present in the sample.The presence of a second amplified sequence can be determined byquantitating the amount of DNA at the start and the end of the secondamplification reaction. Methods for quantitating DNA are well known andinclude determining the optical density at 260 (OD₂₆₀), and preferablyadditionally determining the ratio of the optical density at 260 to theoptical density at 280 (OD₂₆₀ /OD₂₈₀) to determine the amount of DNA incomparison to protein in the sample.

Preferably, the first amplification will contain sufficient primer foronly a limited number of primer extension cycles, e.g. less than 15,preferably about 10 to 12 cycles, so that the amount of amplifiedsequence produced by the process is sufficient for the secondamplification but does not interfere with a determination of whetheramplification occurred with the second primer pair. Alternatively, theamplification reaction can be continued for additional cycles andaliquoted to provide appropriate amounts of DNA for one or more secondamplification reactions. Approximately 100 to 150 ng of each primer ofthe second primer pair is added to the amplification reaction mixture.The second set of primers is preferably added following the initialcycles with the first primer pair. The amount of the first primer paircan be limited in comparison to the second primer pair so that,following addition of the second pair, substantially all of theamplified sequences will be produced by the second pair.

As stated previously, the DNA can be quantitated to determine whether anamplified sequence was produced in the second amplification. If proteinin the reaction mixture interferes with the quantitation (usually due tothe presence of the polymerase), the reaction mixture can be purified,as by using a 100,000 MW cut off filter. Such filters are commerciallyavailable from Millipore and from Centricon.

Analysis of the Amplified DNA Sequence

As discussed previously, the method used to analyze the amplified DNAsequence to characterize the allele(s) present in the sample DNA dependson the genetic variation in the sequence. When distinctions betweenalleles include primer-defined length polymorphisms, the amplifiedsequences are separated based on length, preferably using gel orcapillary electrophoresis. When using probe hybridization for analysis,the amplified sequences are reacted with labeled probes. When theanalysis is based on RFLP fragment patterns, the amplified sequences aredigested with one or more restriction endonucleases to produce a digestand the resultant fragments are separated based on length, preferablyusing gel or capillary electrophoresis. When the only variationencompassed by the amplified sequence is a sequence variation that doesnot result in a change in length or a change in a restriction site andis unsuitable for detection by a probe, the amplified DNA sequences aresequenced.

Procedures for each step of the various analytical methods are wellknown and are described below.

Production of RFLP Fragment Patterns

Restriction endonucleases

A restriction endonuclease is an enzyme that cleaves or cuts DNAhydrolytically at a specific nucleotide sequence called a restrictionsite. Endonucleases that produce blunt end DNA fragments (hydrolysis ofthe phosphodiester bonds on both DNA strands occur at the same site) aswell as endonucleases that produce sticky ended fragments (thehydrolysis sites on the strands are separated by a few nucleotides fromeach other) can be used.

Restriction enzymes are available commercially from a number of sourcesincluding Sigma Pharmaceuticals, Bethesda Research Labs,Boehringer-Manheim and Pharmacia. As stated previously, a restrictionendonuclease used in the present invention cleaves an amplified DNAsequence of this invention to produce a digest comprising a set offragments having distinctive fragment lengths. In particular, thefragments for one allele of a locus differ in size from the fragmentsfor other alleles of the locus. The patterns produced by separation andvisualization of the fragments of a plurality of digests are sufficientto distinguish each allele of the locus. More particularly, theendonucleases are chosen so that by using a plurality of digests of theamplified sequence, preferably fewer than five, more preferably two orthree digests, the alleles of a locus can be distinguished.

In selecting an endonuclease, the important consideration is the numberof fragments produced for amplified sequences of the various alleles ofa locus. More particularly, a sufficient number of fragments must beproduced to distinguish between the alleles and, if required, to providefor individuality determinations. However, the number of fragments mustnot be so large or so similar in size that a pattern that is notdistinguishable from those of other haplotypes by the particulardetection method is produced. Preferably, the fragments are ofdistinctive sizes for each allele. That is, for each endonuclease digestof a particular amplified sequence, the fragments for an allelepreferably differ from the fragments for every other allele of the locusby at least 10, preferably 20, more preferably 30, most preferably 50 ormore nucleotides.

One of ordinary skill can readily determine whether an endonucleaseproduces RFLP fragments having distinctive fragment lengths. Thedetermination can be made experimentally by cleaving an amplifiedsequence for each allele with the designated endonuclease in theinvention method. The fragment patterns can then be analyzed.Distinguishable patterns will be readily recognized by determiningwhether comparison of two or more digest patterns is sufficient todemonstrate characteristic differences between the patterns of thealleles.

The number of digests that need to be prepared for any particularanalysis will depend on the desired information and the particularsample to be analyzed. Since HLA analyses are used for a variety ofpurposes ranging from individuality determinations for forensics andpaternity to tissue typing for transplantation, the HLA complex will beused as exemplary.

A single digest may be sufficient to determine that an individual cannotbe the person whose blood was found at a crime scene. In general,however, where the DNA samples do not differ, the use of two to threedigests for each of two to three HLA loci will be sufficient formatching applications (forensics, paternity). For complete HLA typing,each locus needs to be determined.

In a preferred embodiment, sample HLA DNA sequences are divided intoaliquots containing similar amounts of DNA per aliquot and are amplifiedwith primer pairs (or combinations of primer pairs) to produce amplifiedDNA sequences for a number of HLA loci. Each amplification mixturecontains only primer pairs for one HLA locus. The amplified sequencesare preferably processed concurrently, so that a number of digest RFLPfragment patterns can be produced from one sample. In this way, the HLAtype for a number of alleles can be determined simultaneously.

Alternatively, preparation of a number of RFLP fragment patternsprovides additional comparisons of patterns to distinguish samples forforensic and paternity analyses where analysis of one locus frequentlyfails to provide sufficient information for the determination when thesample DNA has the same allele as the DNA to which it is compared.

Production of RFLP fragments

Following amplification, the amplified DNA sequence is combined with anendonuclease that cleaves or cuts the amplified DNA sequencehydrolytically at a specific restriction site. The combination of theendonuclease with the amplified DNA sequence produces a digestcontaining a set of fragments having distinctive fragment lengths. U.S.Pat. No. 4,582,788 (to Erlich, issued Apr. 15, 1986) describes an HLAtyping method based on restriction length polymorphism (RFLP). Thatpatent is incorporated herein by reference in its entirety.

In a preferred embodiment, two or more aliquots of the amplificationreaction mixture having approximately equal amounts of DNA per aliquotare prepared. Conveniently about 5 to about 10 μl of a 100 μl reactionmixture is used for each aliquot. Each aliquot is combined with adifferent endonuclease to produce a plurality of digests. In this way,by using a number of endonucleases for a particular amplified DNAsequence, locus-specific combinations of endonucleases that distinguisha plurality of alleles of a particular locus can be readily determined.Following preparation of the digests, each of the digests can be used toform RFLP patterns. Preferably, two or more digests can be pooled priorto pattern formation.

Alternatively, two or more restriction endonucleases can be used toproduce a single digest. The digest differs from one where each enzymeis used separately and the resultant fragments are pooled sincefragments produced by one enzyme may include one or more restrictionsites recognized by another enzyme in the digest. Patterns produced bysimultaneous digestion by two or more enzymes will include morefragments than pooled products of separate digestions using thoseenzymes and will be more complex to analyze.

Furthermore, one or more restriction endonucleases can be used to digesttwo or more amplified DNA sequences. That is, for more completeresolution of all the alleles of a locus, it may be desirable to produceamplified DNA sequences encompassing two different regions. Theamplified DNA sequences can be combined and digested with at least onerestriction endonuclease to produce RFLP patterns.

The digestion of the amplified DNA sequence with the endonuclease can becarried out in an aqueous solution under conditions favoringendonuclease activity. Typically the solution is buffered to a pH ofabout 6.5 to 8.0. Mild temperatures, preferably about 20° C. to about45° C., more preferably physiological temperatures (25° to 40° C.), areemployed. Restriction endonucleases normally require magnesium ions and,in some instances, cofactors (ATP and S-adenosyl methionine) or otheragents for their activity. Therefore, a source of such ions, forinstance inorganic magnesium salts, and other agents, when required, arepresent in the digestion mixture. Suitable conditions are described bythe manufacturer of the endonuclease and generally vary as to whetherthe endonuclease requires high, medium or low salt conditions foroptimal activity.

The amount of DNA in the digestion mixture is typically in the range of1% to 20% by weight. In most instances 5 to 20 μg of total DNA digestedto completion provides an adequate sample for production of RFLPfragments. Excess endonuclease, preferably one to five units/μg DNA, isused.

The set of fragments in the digest is preferably further processed toproduce RFLP patterns which are analyzed. If desired, the digest can bepurified by precipitation and resuspension as described by Kan et al,PNAS 75:5631-5635 (1978), prior to additional processing. That articleis incorporated herein by reference in its entirety.

Once produced, the fragments are analyzed by well known methods.Preferably, the fragments are analyzed using electrophoresis. Gelelectrophoresis methods are described in detail hereinafter. Capillaryelectrophoresis methods can be automated (as by using Model 207Aanalytical capillary electrophoresis system from Applied Biosystems ofFoster City, Calif.) and are described in Chin et al, AmericanBiotechnology Laboratory News Edition, December, 1989.

Electrophoretic Separation of DNA Fragments

Electrophoresis is the separation of DNA sequence fragments contained ina supporting medium by size and charge under the influence of an appliedelectric field. Gel sheets or slabs, e.g. agarose, agarose-acrylamide orpolyacrylamide, are typically used for nucleotide sizing gels. Theelectrophoresis conditions affect the desired degree of resolution ofthe fragments. A degree of resolution that separates fragments thatdiffer in size from one another by as little as 10 nucleotides isusually sufficient. Preferably, the gels will be capable of resolvingfragments which differ by 3 to 5 nucleotides. However, for some purposes(where the differences in sequence length are large), discrimination ofsequence differences of at least 100 nt may be sufficiently sensitivefor the analysis.

Preparation and staining of analytical gels is well known. For example,a 3% Nusieve 1% agarose gel which is stained using ethidium bromide isdescribed in Boerwinkle et al, PNAS, 86:212-216 (1989). Detection of DNAin polyacrylamide gels using silver stain is described in Goldman et al,Electrophoresis, 3:24-26 (1982); Marshall, Electrophoresis, 4:269-272(1983); Tegelstrom, Electrophoresis, 7:226-229 (1987); and Allen et al,BioTechniques 7:736-744 (1989). The method described by Allen et al,using large-pore size ultrathin-layer, rehydratable polyacrylamide gelsstained with silver is preferred. Each of those articles is incorporatedherein by reference in its entirety.

Size markers can be run on the same gel to permit estimation of the sizeof the restriction fragments. Comparison to one or more controlsample(s) can be made in addition to or in place of the use of sizemarkers. The size markers or control samples are usually run in one orboth the lanes at the edge of the gel, and preferably, also in at leastone central lane. In carrying out the electrophoresis, the DNA fragmentsare loaded onto one end of the gel slab (commonly called the "origin")and the fragments separate by electrically facilitated transport throughthe gel, with the shortest fragment electrophoresing from the origintowards the other (anode) end of the slab at the fastest rate. Anagarose slab gel is typically electrophoresed using about 100 volts for30 to 45 minutes. A polyacrylamide slab gel is typically electrophoresedusing about 200 to 1,200 volts for 45 to 60 minutes

After electrophoresis, the gel is readied for visualization. The DNAfragments can be visualized by staining the gel with a nucleicacid-specific stain such as ethidium bromide or, preferably, with silverstain, which is not specific for DNA. Ethidium bromide staining isdescribed in Boerwinkle et al, supra. Silver staining is described inGoldman et al, supra, Marshall, supra, Tegelstrom, supra, and Allen etal, supra.

Probes

Allele-specific oligonucleotides or probes are used to identify DNAsequences which have regions that hybridize with the probe sequence. Theamplified DNA sequences defined by a locus-specific primer pair can beused as probes in RFLP analyses using genomic DNA. U.S. Pat. No.4,582,788 (to Erlich, issued Apr. 15, 1986) describes an exemplary HLAtyping method based on analysis of RFLP patterns produced by genomicDNA. The analysis uses cDNA probes to analyze separated DNA fragments ina Southern blot type of analysis. As stated in the patent"[C]omplementary DNA probes that are specific to one (locus-specific) ormore (multilocus) particular HLA DNA sequences involved in thepolymorphism are essential components of the hybridization step of thetyping method" (col. 6, 1. 3-7).

The amplified DNA sequences of the present method can be used as probesin the method described in that patent or in the present method todetect the presence of an amplified DNA sequence of a particular allele.More specifically, an amplified DNA sequence having a known allele canbe produced and used as a probe to detect the presence of the allele insample DNA which is amplified by the present method.

Preferably, however, when a probe is used to distinguish alleles in theamplified DNA sequences of the present invention, the probe has arelatively short sequence (in comparison to the length of the amplifiedDNA sequence) which minimizes the sequence homology of other alleles ofthe locus with the probe sequence. That is, the probes will correspondto a region of the amplified DNA sequence which has the largest numberof nucleotide differences from the amplified DNA sequences of otheralleles produced using that primer pair.

The probes can be labelled with a detectable atom, radical or ligandusing known labeling techniques. Radiolabels, usually ³² P, aretypically used. The probes can be labeled with ³² P by nick translationwith an α-³² P-dNTP (Rigby et al., J. Mol. Biol., 113:237 (1977)) orother available procedures to make the locus-specific probes for use inthe methods described in the patent. The probes are preferably labeledwith an enzyme, such as hydrogen peroxidase. Coupling enzyme labels tonucleotide sequences are well known. Each of the above references isincorporated herein by reference in its entirety.

The analysis method known as "Southern blotting" that is described bySouthern, J. Mol. Biol., 98:503-517 (1975) is an analysis method thatrelies on the use of probes. In Southern blotting the DNA fragments areelectrophoresed, transferred and affixed to a support that binds nucleicacid, and hybridized with an appropriately labeled cDNA probe. Labeledhybrids are detected by autoradiography, or preferably, use of enzymelabels.

Reagents and conditions for blotting are described by Southern, supra;Wahl et al, PNAS 6:3683-3687 (1979); Kan et al, PNAS, supra, U.S. Pat.No. 4:302,204 and Molecular Cloning: A Laboratory Manual by Maniatis etal, Cold Spring Harbor Laboratory 1982. After the transfer is completethe paper is separated from the gel and is dried. Hybridization(annealing) of the resolved single stranded DNA on the paper to an probeis effected by incubating the paper with the probe under hybridizingconditions. See Southern, supra; Kan et al, PNAS, supra and U.S. Pat.No. 4,302,204, col 5, line 8 et seq. Complementary DNA probes specificfor one allele, one locus (locus-specific) or more are essentialcomponents of the hybridization step of the typing method.Locus-specific probes can be made by the amplification method forlocus-specific amplified sequences, described above. The probes are madedetectable by labeling as described above.

The final step in the Southern blotting method is identifying labeledhybrids on the paper (or gel in the solution hybridization embodiment).Autoradiography can be used to detect radiolabel-containing hybrids.Enzyme labels are detected by use of a color development system specificfor the enzyme. In general, the enzyme cleaves a substrate, whichcleavage either causes the substrate to develop or change color. Thecolor can be visually perceptible in natural light or a fluorochromewhich is excited by a known wavelength of light.

Sequencing

Genetic variations in amplified DNA sequences which reflect allelicdifference in the sample DNA can also be detected by sequencing theamplified DNA sequences. Methods for sequencing oligonucleotidesequences are well known and are described in, for example, MolecularCloning: A Laboratory Manual by Maniatis et al, Cold Spring HarborLaboratory 1982. Currently, sequencing can be automated using a numberof commercially available instruments.

Due to the amount of time currently required to obtain sequencinginformation, other analysis methods, such as gel electrophoresis of theamplified DNA sequences or a restriction endonuclease digest thereof arepreferred for clinical analyses.

Kits

As stated previously, the kits of this invention comprise one or more ofthe reagents used in the above described methods. In one embodiment, akit comprises at least one genetic locus-specific primer pair in asuitable container. Preferably the kit contains two or morelocus-specific primer pairs. In one embodiment, the primer pairs are fordifferent loci and are in separate containers. In another embodiment,the primer pairs are specific for the same locus. In that embodiment,the primer pairs will preferably be in the same container when specificfor different alleles of the same genetic locus and in differentcontainers when specific for different portions of the same allelesequence. Sets of primer pairs which are used sequentially can beprovided in separate containers in one kit. The primers of each pair canbe in separate containers, particularly when one primer is used in eachset of primer pairs. However, each pair is preferably provided at aconcentration which facilitates use of the primers at the concentrationsrequired for all amplifications in which it will be used.

The primers can be provided in a small volume (e.g. 100 μl) of asuitable solution such as sterile water or Tris buffer and can befrozen. Alternatively, the primers can be air dried.

In another embodiment, a kit comprises, in separate containers, two ormore endonucleases useful in the methods of this invention. The kit willpreferably contain a locus-specific combination of endonucleases. Theendonucleases can be provided in a suitable solution such as normalsaline or physiologic buffer with 50% glycerol (at about -20° C.) tomaintain enzymatic activity.

The kit can contain one or more locus-specific primer pairs togetherwith locus-specific combinations of endonucleases and may additionallyinclude a control. The control can be an amplified DNA sequence definedby a locus-specific primer pair or DNA having a known HLA type for alocus of interest.

Additional reagents such as amplification buffer, digestion buffer, aDNA polymerase and nucleotide triphosphates can be provided separatelyor in the kit. The kit may additionally contain gel preparation andstaining reagents or preformed gels.

Analyses of exemplary genetic loci are described below.

Analysis of HLA Type

The present method of analysis of genetic variation in an amplified DNAsequence to determine allelic difference in sample DNA can be used todetermine HLA type. Primer pairs that specifically amplify genomic DNAassociated with one HLA locus are described in detail hereinafter. In apreferred embodiment, the primers define a DNA sequence that containsall exons that encode allelic variability associated with the HLA locustogether with at least a portion of one of the adjacent intronsequences. For Class I loci, the variable exons are the second and thirdexons. For Class II loci, the variable exon is the second exon. Theprimers are preferably located so that a substantial portion of theamplified sequence corresponds to intron sequences.

The intron sequences provide restriction sites that, in comparison tocDNA sequences, provide additional information about the individual;e.g., the haplotype. Inclusion of exons within the amplified DNAsequences does not provide as many genetic variations that enabledistinction between alleles as an intron sequence of the same length,particularly for constant exons. This additional intron sequenceinformation is particularly valuable in paternity determinations and inforensic applications. It is also valuable in typing for transplantmatching in that the variable lengths of intron sequences included inthe amplified sequence produced by the primers enables a distinction tobe made between certain heterozygotes (two different alleles) andhomozygotes (two copies of one allele).

Allelic differences in the DNA sequences of HLA loci are illustratedbelow. The tables illustrate the sequence homology of various allelesand indicate exemplary primer binding sites. Table 1 is an illustrationof the alignment of the nucleotides of the Class I A2, A3, Ax, A24(formerly referred to as A9), B27, B58 (formerly referred to as B17),C1, C2 and C3 allele sequences in intervening sequence (IVS) I and III.(The gene sequences and their numbering that are used in the tables andthroughout the specification can be found in the Genbank and/or EuropeanMolecular Biology Laboratories (EMBL) sequence databanks. Thosesequences are incorporated herein by reference in their entirety.)Underlined nucleotides represent the regions of the sequence to whichexemplary locus-specific or Class I-specific primers bind.

Table 2 illustrates the alignment of the nucleotides in IVS I and II ofthe DQA3 (now DQA1 0301), DQA1.2 (now DQA1 0102) and DQA4.1 (now DQA10501) alleles of the DQA1 locus (formerly referred to as the DR4, DR6and DR3 alleles of the DQA1 locus, respectively). Underlined nucleotidesrepresent the regions of the sequence to which exemplary DQA1locus-specific primers bind.

Table 3 illustrates the alignment of the nucleotides in IVS I, exon 2and IVS II of two individuals having the DQw1_(V) allele (designatedhereinafter as DQw1_(V) a and DQw1_(V) b for the upper and lowersequences in the table, respectively), the DQw2 and DQw8 alleles of theDQB1 locus. Nucleotides indicated in the DQw1_(V) b, DQw2 and DQw8allele sequences are those which differ from the DQw1_(V) a sequence.Exon 2 begins and ends at nt 599 and nt 870 of the DQw1_(V) a allelesequence, respectively. Underlined nucleotides represent the regions ofthe sequence to which exemplary DQB1 locus-specific primers bind.

Table 4 illustrates the alignment of the nucleotides in IVS I, exon 2and IVS II of the DPB4.1, DPB9, New and DPw3 alleles of the DPB1 locus.Nucleotides indicated in the DPB9, New and DPw3 allele sequences arethose which differ from the DPB4.1 sequence. Exon 2 begins and ends atnt 7644 and nt 7907 of the DPB4.1 allele sequence, respectively.Underlined nucleotides represent the regions of the sequence to whichexemplary DPB1 locus-specific primers bind.

    TABLE 1      Class I Seq C1 1 GATTACCAATATTGTGCGACCTACTGTATCAATAAAC C2 1 T  C1 38    A    A     AAAGAAACTGGTCTCTATGAGAATCTCTACCTGGTGCTTTCAGACAA C2 38 GG                 A    G      C1 88 CACTTCACCGTTTAAAGAGAAAACTCCTGACTCTACACGTCCATTCCC C2 88  B27 1     T    C     GAGCGCATCAAGTTCTCCGTG C1 138 AGGGCGAGCTCACTGTCTGGCAGCAAGTTCCCC ATGGTCGAGTT    T      C2 138 T--  A2 1 AAGCTTACTCTCTGGCACCAAACTCCATGGGATGATTTTTCCTTCCTAG B27     32 ATCAGTTTCCCT C1 188 TACAAGAGTCCAAGGGGAGAGGTAAGTGTCCTTTATTTTGCTGGATGTAG      C2 187  A2 50 AAGAGTCCAGGTGGACAGGTAAGGAGTGGGAGTCAGGGAGTC B27 44         T    C     ACACAAGAAGGAGAGGTAAGGAGTGAGAGGCAGGGAGTC C1 238 TTTAATATTACCTGAGGTAAGGTAAG     GCAA AGAGTGGGAGGCAGGGAGTC C2 237 C--G                                    A    A      A2 98 CAGTTCCAGGGACAGAGATTACGGGATATGAAAGGAGAGGGACGGGGCCCAT B27 91       A    G     CAGTTCGGACAGGGATTCCAGGAGGAGAAGTGAAGGGGAAGCGGGTGGGC C1 288 CAGTTCAGGGACGGG     GATTCCAGGAGAAGTGAAGGGGAAGGGGCTGGGCG C2 288 --      A2 149 GCCGAGGGTTTCTCCCTTGTTTCTCAGACAGCTCTTGGGCCAAGAC B27 141 GCCACTGGGG     GTCTCTCCCTGGTTTCCACAGACAGATCCTTGTGCCGGAC C1 338 CAGCCTGGGGGTCTCTCCCTGGTTTC    C     ACAGATCCTTGGCCAGGAC C2 337 ----GG                                        C    A      A2 195 TCAGGGAGACATTGAGACAGAGCGCTTGGCAAAGCAGAGGGGTCAGGG B27 191     TCAGGCAGACAGTGTGACAAAGAGGCTGGTGTAGGAGAAGAGGGATCAGG C1 388 TCAGGCACACAGTGTG     ACAAAGATGCTTGGTGTAGGAGAAGAGGGATCAG C2 387 G      A2 246 CGAAGTCCAGGGCCCCAGGCGTTGGCTCTCAGGGTCTCAGGCCCCGAAGG A3 1 Ax 1 A24     1 -- B27 241 AC GAACGTCCAAGGCCCCGGGCGCGGTCTCAGGGTCTCAGGCTCCGAGAG C1 438  C    G     AAAGTCCCAGGTCCCGGGCGGGGTTCTCAGGGTCTCAGGCTCCAAGGG C2 438 --A  A2 296     CGGTGTATGGATTGGGGAGTCCCAGCCTTGGGGATTCCCCAACTCCGCAGTT A3 9 TA-- Ax 9 TGGC     A24 11 ----T B27 291 CCTTGTCTGCATTGGGGAGGCGCACAGTTGGGGTTCCCCACTCCCACGAGTT      C1 488      ##STR1##      C2 488  A2 348 TCTTTTCTCCCTCTCCCAACCTATGTAGGGTCCTTCTTCCTGGATACTCAC A3     60 CTGCAG Ax 61 C------AGCACC A24 61 TG---- B27 344 TCACTTCTTCTCCCAACCTATG     TCGGGTCCTTCTTCCAGGATACTCGT C1 538 GTTCACTTCTTCTCCCAACCTGCGTCGGGTCCTTCTTCCT    G     AATACTCAT C2 538 TA C3 1 TGG      A2 399 GAC GCGGACCCAGTTCTCACTCCCATTGGGTGTCGGGTTTCCAGAGAAGC A3 114 Ax     109 AATCA--T A24 111 G B27 392 GACGCGTCCCCATTTCCACTCCCATTGGGTGTCGGGTGTCTAG     AGAAGC B58 1 C1 588 GACGCGTCCCCAATTCCCACTCCCATTGGGTGTCGGGTTCTAGAAGC C2     589 --AG C3 36 --ACCNNG  A2 449 CAATCAGTGTCGTCGCGGTCGCGGTTCTAAAGTCCGCACG     A3 164 TC Ax 159 GCCCC A24 161 AT B27 442 CAA TCAGTGTCGCCGGGGTCCCAGTTCTAAA    G     TCCCCACG B58 12 C1 635      ##STR2##      C2 637 C C3 87 GGG      A2 489     ##STR3##      A3 204 TCGTGGAGACCAGGC Ax 199 TG A24 201 B27 482      ##STR4##      B58 52 C1 675 CACCCACC CGGACTCAGAATCTCCTCAGACGCCGAGATGCGG C2 677 G C3     127    1st EXON A2 532 GTCATGGCGCCCCGAACCCTCGTCCTGCTACTCTCGGGGGCTC A3     262 CC Ax 242 CCGAC A24 244 GC B27 524      ##STR5##      B58 94 G C1 717      ##STR6##      C2 719 C3 169 G      A2 574     ##STR7##      A3 305 Ax 285 C A24 287 A B27 567 TGGCCCTGACCGAGACCTGGGCTG B58 137 C C1     760 TGGCCCTGACCGAGACCTGGGCCT C2 762 C3 212 G                             C    G        IVS1 A2 599 GTGAGTGCGGGGTGGAGGGAAACG GCCTCTGTGGGGAGAAGCAACGGGCCG A3     329 CACCGT Ax 309 ATCT--G------------GNGGCG A24 311 TCGCCGCG B27 591     G     GTGATGCGGGGTCAGGCAGGGAAATGGCCTCTGTGGGGAGGAGCGAGGGGACG B58 161 G--C C1     784 GTGAGTGCGGGGTTGGGAGGGAAACGGCCTCTGCGGAGAGGAACGA GGTGCCCG C2 786 GG C3     236 TTGG      A2 652     ##STR8##      A3 383 GGC Ax 357 CGTAGA A24 367 A B27 645 CAGGCGGGGGCGCAGGACCCGGGGAGCCG     CGCCGGGAGGAGGGTCGGGCGGGTCTCAG B58 215 TA C1 838 CCCGGCAGGCGCAGGACCCGGGGAG     CCGCGCAGGGAGGAGGGTCGGGCGGGTCTCAG C2 840 GG--AGC C3 291 GGAG  A2 711      ##STR9##      A3 442 G--C Ax 417 TCCT A24 426 B27 703 CCCCT CCTCGCCCCCAG B58 273 C1     895 CCCCTCCTCGCCCCCAG C2 898 T C3 351 --        IVS3 A2 1515     ##STR10##      A3 1245 -- Ax 1222 CACA-- A24 1228 G B27 1508 GTACCAGGGGCAGTGGGGAGCCTTCCC    C     GTCGCCGGGGATGGCCTCCC B58 1082 C1 1704 GTACCAGGGGCAGTGGGGAGCCTTCCCCATCTCCCG     TAGATCTCCCGGCATGGCCTCCC C2 1705 TG C3 1155 --TG                          A    T      A2 1574 ACAAGGAGGGGAGACAGGACCAACACTAGAATATCGCCCTCCCTCTGGT A3 1303     CCGATT Ax 1280 AAAT A24 1287 C B27 1567 ACGAGAAGAGGAGGAAAATGGGATCAGCGCTAG     AATGTCGCCCTCCCTTGAAT B58 1141 C1 1763 ACGAGGAGGGGAGGAAAATGGGATCAGCGCTAGAAT    A     CCCTCCCTGAAAT C2 1764 C3 1213      A2 1627     ##STR11##      A3 1356 TTTT--GAG Ax 1333 TT---------------------- A24 1341 T B27 1620     GGAGAATGGCATGAGTTTTCCTGAGTTTC B58 1194 C1 1816 GGAGAATGGGATGAGTTTTCCTGAGTT    T      C2 1817 C3 1266      A2 1678     ##STR12##      A3 1406 TGAA--G-- Ax 1372 G--GG-- A24 1392 C B27 1649 CTCTGAGGGCCCCCTCTTC    T     GACAATTAAGGGATGACGTC TCTGAGGAA B58 1223 C1 1845 CTCTGAGGGCCCCCTCTGCTCTCTAG     GACAATTAAGGGATGAAGTCCTTGAGGAA C2 1846 C3 1295 GA                         T    C      A2 1733 ATGACGGGAAGACGAAATACTGATGAGTGGTTCCCTTTGACAC A3 1460 GTTGTGG Ax     1426 ATGAAGAG A24 1447 AC B27 1704 ATGGAGGGGAAGACAGTCCCTAGAATACTGATCAGGGG     TCCCCTTTGACCC B58 1278 C1 1900 ATGGAGGGGAAGACAGTCCCTGGAATACTGATCAGGGGTCCCC    T     ACCA C2 1901 C3 1351 A      A2 1783     ##STR13##      A3 1510 ------CGAG Ax 1477 ------TC A24 1497 ------CA B27 1755 CTGCAGCAG     CCTTGGGAACCGTGACTTTTCCTCTCAGGCCTTGTTCACAGC B58 1329 TT C1 1951      ##STR14##      C2 1952 C3 1411 -------------- --      A2 1837 TTCACACTCAATGTGTGTGGGGGTCTGAGTCCAGCACTTCTGAGTCCTTCAGCC A3 1560     C Ax 1528 CC-------------------------- A24 1547 C B27 1806      ##STR15##      B58 1380 C1 2013      ##STR16##      C2 2014 C3 1464 C      A2 1891     ##STR17##      A3 1614 TCA------------------------ Ax 1567 T A24 1600 A----------------     -------- B27 1860      ##STR18##      B58 1434 C1 2067 TCCACTCAGGTCAGGACCAGAAGTCGCTGTTCCTCCCTCAGAGACTAGAACTTTCC    A     AATAG C2 2068 C3 1518                                                    C    T      A2 1955 GAGATTATCCCAGGTGCCTGTGTCCAGGCTGGTGTCTGGGTTTGCTCCCTTCCCCA A3     1664 ---- Ax 1632 TTCTT A24 1650 ------AATG B27 1925      ##STR19##      B58 1499 C1 2132 GAGATTATCCCAGGTGCCTGTGTCCAGGCTGGCGTCTGGGTTCTGTGCCCCCTTCC    C      C2 2133 C3 1583                                                         G      A2 2014 TCCCAGGTGTCCTGTCCATTCTCAA GATAGCCACATGTGTGCTGGAGAGTGTCCCATG A3     1721 GGCT Ax 1691 CTCAAGCT A24 1706 GCAT B27 1983      ##STR20##      B58 1557 A C1 2191      ##STR21##      C2 2192 A C3 1642 G  A2 2073 ACAGATCGAAAATGCCTGAATGATCTGACTCTTCCTGACAG      2113 A3 1780 GCTTCT 1820 Ax 1750 GCTTTTCT 1791 A24 1765 GGCAAAA---------     ---------------------------CT 1784 B27 2042      ##STR22##      2083 B58 1616  1656 C1 2250 AGAGATACAAAGTGTCTGAATTTTCTGACTCTTCCCGTCAG     2290 C2 2251 G 2292 C3 1701  1741

                                      TABLE 2                                     __________________________________________________________________________    DQA1                                                                              Seq                                                                       A3   1                                                                                ##STR23##                                                             A1.2                                                                               1 SGA                                                                    A4.1                                                                               1 CGAACG                                                                 A3   61                                                                              TTTGTTATTAACTGATGAAAGAATTAAGTGAAAGATAAACCTTAGGAAGCAGAGGGAAGT           A1.2                                                                               61                                                                              CATCC                                                                  A4.1                                                                               61                                                                              GTCA                                                                   A3  121                                                                              TAATCTATGACTAAGAAAGTTAAGTACTCTGATAACTCATTCATTCCTTCT                    A1.2                                                                              122                                                                              ACCT AATCCAA                                                           A4.1                                                                              122                                                                              ACCTAACCACAA                                                           A3  172                                                                              TTTGTTCATTTACATTATTTAATCACAAGTCTATGATGTGCCAGGCTCTCAGGAAATA             A1.2                                                                              178                                                                              ATCCA                                                                  A4.1                                                                              178                                                                              AGTCGA                                                                 A3  230                                                                              GTGAAAATTGGCACGCGATATTCTGCCCTTGTGTAGCACACACCGTAGTGGGAAAG               A1.2                                                                              236                                                                              AATGTAG                                                                A4.1                                                                              237                                                                              ACATTGTTA                                                              A3  286                                                                              AAGTGCACTTTTAACCGGACAACTATCAACACGAAGCGGGGAGGAAGCAGGGG                  A1.2                                                                              293                                                                               ATCTA                                                                 A4.1                                                                              294                                                                              ACACATAT                                                               A3  339                                                                              CTGGAAATGTCCACAGACTTTGCCAAAGACAAAGCCCATAATATCTGAAAGTCAG                A1.2                                                                              347                                                                              GAATGT                                                                 A4.1                                                                              348                                                                              TGGTGGT                                                                A3  394                                                                               ##STR24##                                                             A1.2                                                                              403                                                                              GCTCTC                                                                 A4.1                                                                              403                                                                              CTTCATGCCA                                                             A3  450                                                                               ##STR25##                                                             A1.2                                                                              459                                                                              CGT                                                                    A4.1                                                                              462                                                                              CCT                                                                    A3  510                                                                               ##STR26##                                                             A1.2                                                                              519                                                                              TCCCTCC                                                                A4.1                                                                              522                                                                              CCCTCC                                                                 A3  567                                                                              GTGGACCTGGAGAGGAAGGAGACTGTCTGGCAGTTGCCTCTGTTCCGCAGATTTA                A1.2                                                                              576                                                                              CGGGAAAG                                                               A4.1                                                                              579                                                                              GTGTGTCAACA                                                            A3  622                                                                              GAAGATTTGACCCGCAATTTGCACTGACAAACATCGCTGTGC TAAAACATAACTTGA             A1.2                                                                              631                                                                              GTGGGGGGCC                                                             A4.1                                                                              634                                                                              ------C                                                                A3  679                                                                               ##STR27##                                                             A1.2                                                                              688                                                                              AAC                                                                    A4.1                                                                              688                                                                              GTCAA                                                                  A3  740                                                                              CCTTTCTTTACTGATTTATCCCTTTATACCAAGTTTCATTATTTTCTTT                      A1.2                                                                              749                                                                              CTTAAAGCCCGC                                                           A4.1                                                                              749                                                                              CCCA                                                                   A3  789                                                                               ##STR28##                                                             A1.2                                                                              802                                                                              819                                                                    A4.1                                                                              798                                                                              815                                                                    __________________________________________________________________________

                                      TABLE 3                                     __________________________________________________________________________    DQB1 Seq                                                                       1    AAGCTTGTGCTCTTTCCATGAATAAATGTCTCTATCTAGGACTCAGAGGT                            GGTTA                                                                         G                                                                        51   GTAGGTCCTTTCCAACATAGAAGGGAGTGAACCTCAACGGGACTTGGGAG                            TTTT                                                                          CACCTTTTACCAACGTGACAC                                                         ATATCA                                                                  101                                                                                  ##STR29##                                                                    C                                                                             G                                                                       151                                                                                  ##STR30##                                                                    GAG--ATG                                                                      AATCGA                                                                  201   TCCGTTGAACTCTCAGATTTATGTGGATAACTTTATCTCTGAGGTATCCA                            CGGC                                                                          CAGTT                                                                   251   GGAGCTTCATGAAAAATGGGATTTCATGCGAGAACGCCCTGATCCCTCTA                            CGA                                                                           CAGGT                                                                   301                                                                                  ##STR31##                                                                    CAT                                                                           CTCC                                                                    351   CAGGCTCAGGCAGGGACAGGGCTTTCCTCCCTTTCCTGGATGTAGGAAGG                            CGACC                                                                         CGCCC                                                                   401   CAGATTCCAGAAGCCCGCAAAGAAGGCGGGCAGAGCTGGGCAGAGCCGCC                            CGCACCGGG--NNN                                                                GCCGGG                                                                  451   GGGAGGATCCCAGGTCTGGAGCGCCAGGCACGGGCGGGCGGGAACTGGAG                            CGTT                                                                          CAA                                                                     501   GTCGCGCGGGCGGTTCCACAGCTCCAGGCCGGGTCAGGGCGGCGGCTGCG                            T GT                                                                          G                                                                       551                                                                                  ##STR32##                                                                    A--GCA------                                                                  GGGCCGGGGCC                                                             601                                                                                  ##STR33##                                                                    A                                                                       651                                                                                  ##STR34##                                                                    GGAGAATT                                                                      GTA                                                                     701                                                                                  ##STR35##                                                                    ATTTT                                                                         TC                                                                      751                                                                                  ##STR36##                                                                    CCCAAA                                                                        CC                                                                      801                                                                                  ##STR37##                                                                    CGGCTACTA                                                                     ACTACTA                                                                 851   GGATCCTGCAGAGGAGAGGTGAGCTTCGTCGCCCCTCCGTGAGCGCACCC                            G                                                                             CCTCCGG--TTCGCC                                                               CCTCCGGGCCT                                                             901   TTGGCCGGGACCCCGA GTCTCTGTGCCGGGAGGGCGATGGGGGCGAGGTC                           ----------ACAGCAATTC                                                          AGACCGGCGAACC                                                           951   TCTGAAATCTTGAGCCCAGTTCATTCCACCCCAGGGAAAGGAGGCGGCGG                            --                                                                            --C--CGG                                                                      GCTT--CTGC--AA                                                          1001  CGGGGGTGGTGGGGGCAGGTGCATCGGAGGGGCGGGGACCTAGGGCAGAG                            CGGT--CTA                                                               1051  CAGGGGGACAAGCAGAGTTGGCCAGGCTGCCTAGTGTCCCCCCCAGCCTC                            GTATG--T                                                                1101                                                                                 ##STR38##                                                                    C                                                                             CCC--T                                                                  1151                                                                                 ##STR39##                                                                    TA                                                                      1201  CCCAGTGCCCACCCTCTTCCCCTGCCCGCCGGCCTCGCTAGCACTGCCCC                            ATTGCCGG                                                                1251  ACCCAGCAAGGCCCACAGTCGCGCATTCGCCGCAGGAAGCTT1292                                TCG                                                                           GTCTAAAGCCATGAGTGGGAAGCTT                                               __________________________________________________________________________

                                      TABLE 4                                     __________________________________________________________________________    DPB1 Seq                                                                      DPB4.1 7546                                                                           GGGAAGATTTGGGAAGAATCGTTAATAT                                          DPB4.1 7574                                                                            ##STR40##                                                            DPB4.1 7634                                                                           CTCCCCGCAGAGAATTACCTTTTCCAGGGACGGCAGGAATGCTACGCGTTTAATGGGACA          DPB9    GGATGGCATT                                                            New     GGATGGCATT                                                            DPw3                                                                          BPB4.1 7694                                                                           CAGCGCTTCCTGGAGAGATACATCTACAACCGGGAGGAGTTCGCGCGCTTCGACAGCGAC          DPB9    T                                                                     New     T                                                                     DPw3                                                                          DPB4.1 7754                                                                           GTGGGGGAGTT CCGGGCGGTGACGGAGCTGGGGCGGCCTGCTGCGGAGTACTGGAACAGC         DPB9    AAC                                                                   New     AAC                                                                   DPw3                                                                          DPB4.1 7814                                                                           CAGAAGGACATCCTGGAGGAGAAGCGGGCAGTGCCGGACAGGATGTGCAGACACAACTAC          DPB9    GGA                                                                   New     CGA                                                                   DPw3    CGA                                                                   DPB4.1 7874                                                                            ##STR41##                                                            DPB9    AAGG                                                                  New     AAGG                                                                  DPw3    AAGG                                                                  DPB4.1 7934                                                                           CCCAGGGCAGCCCCGCGGGCCCGTGCCCAG                                        __________________________________________________________________________

Primers for HLA loci

Exemplary HLA locus-specific primers are listed below. Each of theprimers hybridizes with at least about 15 consecutive nucleotides of thedesignated region of the allele sequence. The designation of anexemplary preferred primer together with its sequence is also shown. Formany of the primers, the sequence is not identical for all of the otheralleles of the locus. For each of the following preferred primers,additional preferred primers have sequences which correspond to thesequences of the homologous region of other alleles of the locus or totheir complements.

In one embodiment, Class I loci are amplified by using an A, B or Clocus-specific primer together with a Class I locus-specific primer. TheClass I primer preferably hybridizes with IVS III sequences (or theircomplements) or, more preferably, with IVS I sequences (or theircomplements). The term "Class I-specific primer", as used herein, meansthat the primer hybridizes with an allele sequence (or its complement)for at least two different Class I loci and does not hybridize withClass II locus allele sequences under the conditions used. Preferably,the Class I primer hybridizes with at least one allele of each of the A,B and C loci. More preferably, the Class I primer hybridizes with aplurality of, most preferably all of, the Class I allele loci or theircomplements. Exemplary Class I locus-specific primers are also listedbelow.

HLA Primers

A locus-specific primers

allelic location: nt 1735-1757 of A3

designation: SGD009.AIVS3.R2NP

sequence: CATGTGGCCATCTTGAGAATGGA

allelic location: nt 1541-1564 of A2

designation: SGD006.AIVS3.R1NP

sequence: GCCCGGGAGATCTACAGGCGATCA

allelic location: nt 1533-1553 of A2

designation: A2.1

sequence: CGCCTCCCTGATCGCCTGTAG

allelic location: nt 1667-1685 of A2

designation: A2.2

sequence: CCAGAGAGTGACTCTGAGG

allelic location: nt 1704-1717 of A2

designation: A2.3

sequence: CACAATTAAGGGAT

B locus-specific primers

allelic location: nt 1108-1131 of B17

designation: SGD007.BIVS3.R1NP

sequence: TCCCCGGCGACCTATAGGAGATGG

allelic location: nt 1582-1604 of B17

designation: SGD010.BIVS3.R2NP

sequence: CTAGGACCACCCATGTGACCAGC

allelic location: nt 500-528 of B27

designation: B2.1

sequence: ATCTCCTCAGACGCCGAGATGCGTCAC

allelic location: nt 545-566 of B27

designation: B2.2

sequence: CTCCTGCTGCTCTGGGGGGCAG

allelic location: nt 1852-1876 of B27

designation: B2.3

sequence: ACTTTACCTCCACTCAGATCAGGAG

allelic location: nt 1945-1976 of B27

designation: B2.4

sequence: CGTCCAGGCTGGTGTCTGGGTTCTGTGCCCCT

allelic location: nt 2009-2031 of B27

designation: B2.5

sequence: CTGGTCACATGGGTGGTCCTAGG

allelic location: nt 2054-2079 of B27

designation: B2.6

sequence: CGCCTGAATTTTCTGACTCTTCCCAT

C locus-specific primers

allelic location: nt 1182-1204 of C3

designation: SGD008.CIVS3.R1NP

sequence: ATCCCGGGAGATCTACAGGAGATG

allelic location: nt 1665-1687 of C3

designation: SGD011.CIVS3.R2NP

sequence: AACAGCGCCCATGTGACCATCCT

allelic location: nt 499-525 of C1

designation: C2.1

sequence: CTGGGGAGGCGCCGCGTTGAGGATTCT

allelic location: nt 642-674 of C1

designation: C2.2

sequence: CGTCTCCGCAGTCCCGGTTCTAAAGTTCCCAGT

allelic location: nt 738-755 of C1

designation: C2.3

sequence: ATCCTCGTGCTCTCGGGA

allelic location: nt 1970-1987 of C1

designation: C2.4

sequence: TGTGGTCAGGCTGCTGAC

allelic location: nt 2032-2051 of C1

designation: C2.5

sequence: AAGGTTTGATTCCAGCTT

allelic location: nt 2180-2217 of C1

designation: C2.6

sequence: CCCCTTCCCCACCCCAGGTGTTCCTGTCCATTCTTCAGGA

allelic location: nt 2222-2245 of C1

designation: C2.7

sequence: CACATGGGCGCTGTTGGAGTGTCG

Class I loci-specific primers

allelic location: nt 599-620 of A2

designation: SGD005.IIVS1.LNP

sequence: GTGAGTGCGGGGTCGGGAGGGA

allelic location: nt 489-506 of A2

designation: 1.1

sequence: CACCCACCGGGACTCAGA

allelic location: nt 574-595 of A2

designation: 1.2

sequence: TGGCCCTGACCCAGACCTGGGC

allelic location: nt 691-711 of A2

designation: 1.3

sequence: GAGGGTCGGGCGGGTCTCAGC

allelic location: nt 1816-1831 of A2

designation: 1.4

sequence: CTCTCAGGCCTTGTTC

allelic location: nt 1980-1923 of A2

designation: 1.5

sequence: CAGAAGTCGCTGTTCC

DQA1 locus-specific primers

allelic location: nt 23-41 of DQA3

designation: SGD001.DQA1.LNP

sequence: TTCTGAGCCAGTCCTGAGA

allelic location: nt 45-64 of DQA3

designation: DQA3 E1a

sequence: TTGCCCTGACCACCGTGATG

allelic location: nt 444-463 of DQA3

designation: DQA3 E1b

sequence: CTTCCTGCTTGTCATCTTCA

allelic location: nt 536-553 of DQA3

designation: DQA3 E1c

sequence: CCATGAATTTGATGGAGA

allelic location: nt 705-723 of DQA3

designation: DQA3 E1d

sequence: ACCGCTGCTACCAATGGTA

allelic location: nt 789-806 of DQA3

designation: SGD003.DQA1.RNP

sequence: CCAAGAGGTCCCCAGATC

DRA locus-specific primers

allelic location: nt 49-68 of DRA HUMMHDRAM (1183 nt sequence, AccessionNo. K1171)

designation: DRA E1

sequence: TCATCATAGCTGTGCTGATG

allelic location: nt 98-118 of DRA HUMMHDRAM (1183 nt sequence,Accession No. K01177)

designation: DRA 5'E2 (5' indicates the primer is used as the 5' primer)

sequence: AGAACATGTGATCATCCAGGC

allelic location: nt 319-341 of DRA HUMMHDRAM (1183 nt sequence,Accession No. K01171)

designation: DRA 3'E2

sequence: CCAACTATACTCCGATCACCAAT

DRB locus-specific primers

allelic location: nt 79-101 of DRB HUMMHDRC (1153 nt sequence, AccessionNo. K01171)

designation: DRB E1

sequence: TGACAGTGACACTGATGGTGCTG

allelic location: nt 123-143 of DRB HUMMHDRC (1153 nt sequence,Accession No. K01171)

designation: DRB 5'E2

sequence: GGGGACACCCGACCACGTTTC

allelic location: nt 357-378 of DRB HUMMHDRC (1153 nt sequence,Accession No. K01171)

designation: DRB 3'E2

sequence: TGCAGACACAACTACGGGGTTG

DQB1 locus-specific primers

allelic location: nt 509-532 DQB1 DQw1_(v) a

designation: DQB E1

sequence: TGGCTGAGGGCAGAGACTCTCCC

allelic location: nt 628-647 of DQB1 DQw1_(v) a

designation: DQB 5'E2

sequence: TGCTACTTCACCAACGGGAC

allelic location: nt 816-834 of DQB1 DQw1_(v) a

designation: DQB 3,E2

sequence: GGTGTGCACACACAACTAC

allelic location: 124-152 of DQB1 DQw1_(v) a

designation: DQB 5' IVS1a

sequence: AGGTATTTTACCCAGGGACCAAGAGAT

allelic location: nt 314-340 of DQB1 DQw1_(v) a

designation: DQB 5' IVS1b

sequence: ATGTAAAATCAGCCCGACTGCCTCTTC

allelic location: nt 1140-1166 of DQB1 DQw1_(v) a

designation: DQB 3' IVS2

sequence: GCCTCGTGCCTTATGCGTTTGCCTCCT

DPB1 locus-specific primers

allelic location: nt 6116-6136 of DPB1 4.1

designation: DPB E1

sequence: TGAGGTTAATAAACTGGAGAA

allelic location: nt 7604-7624 of DPB1 4.1

designation: DPB 5' IVSI

sequence: GAGAGTGGCGCCTCCGCTCAT

allelic location: nt 7910-7929 of DPB1 4.1

designation: DPB 3' IVS2

sequence: GAGTGAGGGCTTTGGGCCGG

Primer pairs for HLA analyses

It is well understood that for each primer pair, the 5' upstream primerhybridizes with the 5' end of the sequence to be amplified and the 3'downstream primer hybridizers with the complement of the 3' end of thesequence. The primers amplify a sequence between the regions of the DNAto which the primers bind and its complementary sequence including theregions to which the primers bind. Therefore, for each of the primersdescribed above, whether the primer binds to the HLA-encoding strand orits complement depends on whether the primer functions as the 5'upstream primer or the 3' downstream primer for that particular primerpair.

In one embodiment, a Class I locus-specific primer pair includes a ClassI locus-specific primer and an A, B or C locus-specific primer.Preferably, the Class I locus-specific primer is the 5' upstream primerand hybridizes with a portion of the complement of IVS I. In that case,the locus-specific primer is preferably the 3' downstream primer andhybridizes with IVS III. The primer pairs amplify a sequence of about1.0 to about 1.5 Kb.

In another embodiment, the primer pair comprises two locus-specificprimers that amplify a DNA sequence that does not include the variableexon(s). In one example of that embodiment, the 3' downstream primer andthe 5' upstream primer are Class I locus-specific primers that hybridizewith IVS III and its complement, respectively. In that case a sequenceof about 0.5 Kb corresponding to the intron sequence is amplified.

Preferably, locus-specific primers for the particular locus, rather thanfor the HLA class, are used for each primer of the primer pair. Due todifferences in the Class II gene sequences, locusspecific primers whichare specific for only one locus participate in amplifying the DRB, DQA1,DQB and DPB loci. Therefore, for each of the preferred Class II locusprimer pairs, each primer of the pair participates in amplifying onlythe designated locus and no other Class II loci.

Analytical methods

In one embodiment, the amplified sequence includes sufficient intronsequences to encompass length polymorphisms. The primer-defined lengthpolymorphisms (PDLPs) are indicative of the HLA locus allele in thesample. For some HLA loci, use of a single primer pair producesprimer-defined length polymorphisms that distinguish between some of thealleles of the locus. For other loci, two or more pairs of primers areused in separate amplifications to distinguish the alleles. For otherloci, the amplified DNA sequence is cleaved with one or more restrictionendonucleases to distinguish the alleles. The primer-defined lengthpolymorphisms are particularly useful in screening processes.

In anther embodiment, the invention provides an improved method thatuses PCR amplification of a genomic HLA DNA sequence of one HLA locus.Following amplification, the amplified DNA sequence is combined with atleast one endonuclease to produce a digest. The endonuclease cleaves theamplified DNA sequence to yield a set of fragments having distinctivefragment lengths. Usually the amplified sequence is divided, and two ormore endonuclease digests are produced. The digests can be used, eitherseparately or combined, to produce RFLP patterns that can distinguishbetween individuals. Additional digests can be prepared to provideenhanced specificity to distinguish between even closely relatedindividuals with the same HLA type.

In a preferred embodiment, the presence of a particular allele can beverified by performing a two step amplification procedure in which anamplified sequence produced by a first primer pair is amplified by asecond primer pair which binds to and defines a sequence within thefirst amplified sequence. The first primer pair can be specific for oneor more alleles of the HLA locus. The second primer pair is preferablyspecific for one allele of the HLA locus, rather than a plurality ofalleles. The presence of an amplified sequence indicates the presence ofthe allele, which is confirmed by production of characteristic RFLPpatterns.

To analyze RFLP patterns, fragments in the digest are separated by sizeand then visualized. In the case of typing for a particular HLA locus,the analysis is directed to detecting the two DNA allele sequences thatuniquely characterize that locus in each individual. Usually this isperformed by comparing the sample digest RFLP patterns to a patternproduced by a control sample of known HLA allele type. However, when themethod is used for paternity testing or forensics, the analysis need notinvolve identifying a particular locus or loci but can be done bycomparing single or multiple RFLP patterns of one individual with thatof another individual using the same restriction endonuclease andprimers to determine similarities and differences between the patterns.

The number of digests that need to be prepared for any particularanalysis will depend on the desired information and the particularsample to be analyzed. For example, one digest may be sufficient todetermine that an individual cannot be the person whose blood was foundat a crime scene. In general, the use of two to three digests for eachof two to three HLA loci will be sufficient for matching applications(forensics, paternity). For complete HLA haplotyping; e.g., fortransplantation, additional loci may need to be analyzed.

As described previously, combinations of primer pairs can be used in theamplification method to amplify a particular HLA DNA locus irrespectiveof the allele present in the sample. In a preferred embodiment, samplesof HLA DNA are divided into aliquots containing similar amounts of DNAper aliquot and are amplified with primer pairs (or combinations ofprimer pairs) to produce amplified DNA sequences for additional HLAloci. Each amplification mixture contains only primer pairs for one HLAlocus. The amplified sequences are preferably processed concurrently, sothat a number of digest RFLP fragment patterns can be produced from onesample. In this way, the HLA type for a number of alleles can bedetermined simultaneously.

Alternatively, preparation of a number of RFLP fragment patternsprovides additional comparisons of patterns to distinguish samples forforensic and paternity analyses where analysis of one locus frequentlyfails to provide sufficient information for the determination when thesample DNA has the same allele as the DNA to which it is compared.

The use of HLA types in paternity tests or transplantation testing andin disease diagnosis and prognosis is described in Basic & ClinicalImmunology, 3rd Ed (1980) Lange Medical Publications, pp 187-190, whichis incorporated herein by reference in its entirety. HLA determinationsfall into two general categories. The first involves matching of DNAfrom an individual and a sample. This category involves forensicdeterminations and paternity testing. For category 1 analysis, theparticular HLA type is not as important as whether the DNA from theindividuals is related. The second category is in tissue typing such asfor use in transplantation. In this case, rejection of the donated bloodor tissue will depend on whether the recipient and the donor express thesame or different antigens. This is in contrast to first categoryanalyses where differences in the HLA DNA in either the introns or exonsis determinative.

For forensic applications, analysis of the sample DNA of the suspectedperpetrator of the crime and DNA found at the crime scene are analyzedconcurrently and compared to determine whether the DNA is from the sameindividual. The determination preferably includes analysis of at leastthree digests of amplified DNA of the DQA1 locus and preferably also ofthe A locus. More preferably, the determination also includes analysisof at least three digests of amplified DNA of an additional locus, e.g.the DPB locus. In this way, the probability that differences between theDNA samples can be discriminated is sufficient.

For paternity testing, the analysis involves comparison of DNA of thechild, the mother and the putative father to determine the probabilitythat the child inherited the obligate haplotype DNA from the putativefather. That is, any DNA sequence in the child that is not present inthe mother's DNA must be consistent with being provided by the putativefather. Analysis of two to three digests for the DQA1 and preferablyalso for the A locus is usually sufficient. More preferably, thedetermination also includes analysis of digests of an additional locus,e.g. the DPB locus.

For tissue typing determinations for transplantation matching, analysisof three loci (HLA A, B, and DR) is often sufficient. Preferably, thefinal analysis involves comparison of additional loci including DQ andDP.

Production of RFLP fragment patterns

The following table of exemplary fragment pattern lengths demonstratesdistinctive patterns. For example, as shown in the table, BsrI cleavesA2, A3 and A9 allele amplified sequences defined by primersSGD005.IIVS1.LNP and SGD009.AIVS3.R2NP into sets of fragments with thefollowing numbers of nucleotides (740, 691), (809, 335, 283) and (619,462, 256, 93), respectively. The fragment patterns clearly indicatewhich of the three A alleles is present. The following table illustratesa number of exemplary endonucleases that produce distinctive RFLPfragment patterns for exemplary A allele sequences.

Table 2 illustrates the set of RFLP fragments produced by use of thedesignated endonucleases for analysis of three A locus alleles. For eachendonuclease, the number of nucleotides of each of the fragments in aset produced by the endonuclease is listed. The first portion of thetable illustrates RFLP fragment lengths using the primers designatedSGD009.AIVS3.R2NP and SGD005.IIVS1.LNP which produce the longer of thetwo exemplary sequences. The second portion of the table illustratesRFLP fragment lengths using the primers designated SGD006.AIVS3.R1NP andSGD005.IIVS1.LNP which produce the shorter of the sequences. The thirdportion of the table illustrates the lengths of fragments of a DQA1locus-specific amplified sequence defined by the primers designatedSGD001.DQA1.LNP and SGD003.DQA1.RNP.

As shown in the Table, each of the endonucleases produces acharacteristic RFLP fragment pattern which can readily distinguish whichof the three A alleles is present in a sample.

                                      TABLE 5                                     __________________________________________________________________________    RFLP FRAGMENT PATTERNS                                                        __________________________________________________________________________    A - LONG                           A - SHORT                                  BsrI                                                                              A2   740                                                                              691                  691              254                             A3                                                                              809            335                                                                              283              345                                                                              335                                                                              283                                A9         619                                                                              462      256                                                                              93      619         256                                                                              93                       Cfr101                                                                            A2                                                                              1055     399                                                                              245                                                             A3      473                                                                              399                                                                              247                                                             A9   786   399                                                            DraII                                                                             A2                                                                              698            251   138           295                                                                              251                                                                              210                                                                              138                             A3         369                                                                              315                                                                              251                                                                              247           315   251                                                                              210                                A9   596                                                                              427      251      80 427        251                                                                              210                            FokI                                                                              A2   728   248      151           293                                                                              248      151                                                                              143                                                                              129    51                 A3      515   225                                                                              213                                                                              151                 225                                                                              213                                                                              151                                                                              143                                                                              129    51                 A9                                                                              1004              151      539              151                                                                              146                                                                              129                   GsuI                                                                              A2   868   547         36         868         61 36                           A3                                                                              904         523            904              59                              A9      638      419                                                                              373              414                                                                              373                                                                              178                            HphI                                                                              A2                                                                              1040           239      72 554        339                                   A3      419                                                                              375      218                                                                              163        411                                                                              375   177                                A9   643                                                                              419                                                                              373                    414                                                                              373   178                            MboII                                                                             A2   1011     165                                                                              143                                                                              132                                                       A3      893                                                                              194   143   115                                                    A9                                                                              1349                    51                                              PpumI                                                                             A2                                                                              698            295                                                                              251   138        295                                                                              257   212   69                        A3         369                                                                              364   251                                                                              242        364      251                                                                              210                                                                              72 66                        A9   676                                                                              503         251      503           251                                                                              211                         PssI                                                                              A2                                                                              695            295                                                                              251   138        295                                                                              251                                                                              219   72                           A3         366                                                                              315   251                                                                              242        315   251   207                                                                              72 66                        A9   596                                                                              427         251      427        251   208                                                                              72                       __________________________________________________________________________              DQA1                                                                AluI DQA3    449   335                                                             DQA4.1     338                                                                              332                122                                          DQA1.2        335                                                                              287             123                  52                 CvijI                                                                              DQA3                271       187                                                                              122    99        64                          DQA4.1              277                                                                              219           102   79         55                      DQA1.2                    201        101                                                                              99 80  76     55                 DdeI DQA3 587                                   88     65                          DQA4.1     388                194          89     64                          DQA1.2     395                165          88     65  41                 MboII                                                                              DQA3       366                184                                                                              172              62                          DQA4.1  407                                                                              353                                                                DQA1.2     330                                                                              316                          89                            MnlI DQA3                   214    176                                                                              172           72     43                      DQA4.1           294          179       149           40                      DQA1.2                 216           136                                                                              123                                                                              73  54 44  40                 NlaIII                                                                             DQA3    458         266                        60                             DQA4.1           300                                                                              263                                                                              229                                                    DQA1.2                 223                                                                              190           124                                                                              116 75                        TthIIIII                                                                           DQA3    417            226           141                                      DQA4.1                                                                             426   371                                                                DQA1.2                                                                             428                         148 141       75                        __________________________________________________________________________                                                DQA1                                                              AluI  DQA3                                                                          DQA4.1                                                                        DQA1.2                                                                  CvijI DQA3  34          7                                                           DQA4.1                                                                              36    17    7                                                           DQA1.2                                                                              36 35       7                                                     DdeI  DQA3     30    11 3                                                           DQA4.1                                                                              36       11 3                                                           DQA1.2                                                                              36       11 3                                                     MboII DQA3                                                                          DQA4.1                                                                              32                                                                      DQA1.2                                                                              32 30                                                             MnlI  DQA3  36 23 21 17 10                                                          DQA4.1                                                                              36 33 21                                                                DQA1.2                                                                              36 24 21 15 10 5                                                  NlaIII                                                                              DQA3                                                                          DQA4.1                                                                        DQA1.2                                                                              39 30                                                             TthIIIII                                                                            DQA3                                                                          DQA4.1                                                                        DQA1.2                                  __________________________________________________________________________

Screening Analysis for Genetic Disease

Carriers of genetic diseases and those affected by the disease can beidentified by use of the present method. Depending on the disease, thescreening analysis can be used to detect the presence of one or morealleles associated with the disease or the presence of haplotypesassociated with the disease. Furthermore, by analyzing haplotypes, themethod can detect genetic diseases that are not associated with codingregion variations but are found in regulatory or other untranslatedregions of the genetic locus. The screening method is exemplified belowby analysis of cystic fibrosis (CF).

Cystic fibrosis is an autosomal recessive disease, requiring thepresence of a mutant gene on each chromosome. CF is the most commongenetic disease in Caucasians, occurring once in 2,000 live births. Itis estimated that one in forty Caucasians are carriers for the disease.

Recently a specific deletion of three adjacent basepairs in the openreading frame of the putative CF gene leading to the loss of aphenylalanine residue at position 508 of the predicted 1480 amino acidpolypeptide was reported [Kerem et al, Science 245:1073-1080 (1989)].Based on haplotype analysis, the deletion may account for most CFmutations in Northern European populations (about 68%). A secondmutation is reportedly prevalent in some Southern European populations.Additional data indicate that several other mutations may cause thedisease.

Studies of haplotypes of parents of CF patients (who necessarily haveone normal and one disease-associated haplotype) indicated that thereare at least 178 haplotypes associated with the CF locus. Of thosehaplotypes, 90 are associated only with the disease; 78 are found onlyin normals; and 10 are associated with both the disease and with normals(Kerem et al, supra). The disease apparently is caused by severaldifferent mutations, some in very low frequency in the population. Asdemonstrated by the haplotype information, there are more haplotypesassociated with the locus than there are mutant alleles responsible forthe disease.

A genetic screening program (based on amplification of exon regions andanalysis of the resultant amplified DNA sequence with probes specificfor each of the mutations or with enzymes producing RFLP patternscharacteristic of each mutation) may take years to develop. Such testswould depend on detection and characterization of each of the mutations,or at least of mutations causing about 90 to 95% or more of the cases ofthe disease. The alternative is to detect only 70 to 80% of theCF-associated genes. That alternative is generally consideredunacceptable and is the cause of much concern in the scientificcommunity.

The present method directly determines haplotypes associated with thelocus and can detect haplotypes among the 178 currently recognizedhaplotypes associated with the disease locus. Additional haplotypesassociated with the disease are readily determined through the rapidanalysis of DNA of numerous CF patients by the methods of thisinvention. Furthermore, any mutations which may be associated withnoncoding regulatory regions can also be detected by the method and willbe identified by the screening process.

Rather than attempting to determine and then detect each defect in acoding region that causes the disease, the present method amplifiesintron sequences associated with the locus to determine allelic andsuballelic patterns. In contrast to use of mutation-specific probeswhere only known sequence defects can be detected, new PDLP and RFLPpatterns produced by intron sequences indicate the presence of apreviously unrecognized haplotype.

The same analysis can be performed for phenylalanine hydroxylase locusnutations that cause phenylketonuria and for beta-globin mutations thatcause beta-thalassemia and sickle cell disease and for other loci knownto be associated with a genetic disease. Furthermore, neither themutation site nor the location for a disease gene is required todetermine haplotypes associated with the disease. Amplified intronsequences in the regions of closely flanking RFLP markers, such as areknown for Huntington's disease and many other inherited diseases, canprovide sufficient information to screen for haplotypes associated withthe disease.

Muscular dystrophy (MD) is a sex-linked disease. The disease-associatedgene comprises a 2.3 million basepair sequence that encodes 3,685 aminoacid protein, dystrophin. A map of mutations for 128 of 34 patients withBecker's muscular dystrophy and 160 patients with Duchenne musculardystrophy identified 115 deletions and 13 duplications in the codingregion sequence [Den Dunnen et al, Am. J. Hum. Genet. 45:835-847(1989)]. Although the disease is associated with a large number ofmutations that vary widely, the mutations have a non-random distributionin the sequence and are localized to two major mutation hot spots, DenDunnen et al, supra.

Further, a recombination hot spot within the gene sequence has beenidentified [Grimm et al, Am. J. Hum. Genet. 45:368-372 (1989)].

For analysis of MD, haplotypes on each side of the recombination hotspot are preferably determined. Primer pairs defining amplified DNAsequences are preferably located near, within about 1 to 10 Kbp of thehot spot on either side of the hot spot. In addition, due to the largesize of the gene, primer pairs defining amplified DNA sequences arepreferably located near each end of the gene sequence and mostpreferably also in an intermediate location on each side of the hotspot. In this way, haplotypes associated with the disease can beidentified.

Other diseases, particularly malignancies, have been shown to be theresult of an inherited recessive gene together with a somatic mutationof the normal gene. One malignancy that is due to such "loss ofheterogeneity" is retinoblastoma, a childhood cancer. The loss of thenormal gene through mutation has been demonstrated by detection of thepresence of one mutation in all somatic cells (indicating germ cellorigin) and detection of a second mutation in some somatic cells[Scheffer et al, Am. J. Hum. Genet. 45:252-260 (1989)]. The disease canbe detected by amplifying somatic cell, genomic DNA sequences thatencompass sufficient intron sequence nucleotides. The amplified DNAsequences preferably encompass intron sequences locate near one or moreof the markers described by Scheffer et al, supra. Preferably, anamplified DNA sequence located near an intragenic marker and anamplified DNA sequence located near a flanking marker are used.

An exemplary analysis for CF is described in detail in the examples.Analysis of genetic loci for other monogenic and multigenic geneticdiseases can be performed in a similar manner.

As the foregoing description indicates, the present method of analysisof intron sequences is generally applicable to detection of any type ofgenetic trait. Other monogenic and multigenic traits can be readilyanalyzed by the methods of the present invention. Furthermore, theanalysis methods of the present method are applicable to all eukaryoticcells, and are preferably used on those of plants and animals. Examplesof analysis of BoLA (bovine MHC determinants) further demonstrates thegeneral applicability of the methods of this invention.

This invention is further illustrated by the following specific butnon-limiting examples. Procedures that are constructively reduced topractice are described in the present tense, and procedures that havebeen carried out in the laboratory are set forth in the past tense.

EXAMPLE 1 Forensic Testing

DNA extracted from peripheral blood of the suspected perpetrator of acrime and DNA from blood found at the crime scene are analyzed todetermine whether the two samples of DNA are from the same individual orfrom different individuals.

The extracted DNA from each sample is used to form two replicatealiquots per sample, each aliquot having 1 μg of sample DNA. Eachreplicate is combined in a total volume of 100 μl with a primer pair (1μg of each primer), dNTPs (2.5 mM each) and 2.5 units of Taq polymerasein amplification buffer (50 mM KCl; 10 mM Tris-HCl, pH 8.0; 2.5 mM MgCl₂; 100 μg/ml gelatin) to form four amplification reaction mixtures. Thefirst primer pair contains the primers designated SGD005.IIVS1.LNP andSGD009.AIVS3.R2NP (A locus-specific). The second primer pair containsthe primers designated SGD001.DQA1.LNP and SGD003.DQA1.RNP (DQAlocus-specific). Each primer is synthesized using an Applied Biosystemsmodel 308A DNA synthesizer. The amplification reaction mixtures aredesignated SA (suspect's DNA, A locus-specific primers), SD (suspect'sDNA, DQA1 locus-specific primers), CA (crime scene DNA, A locus-specificprimers) and CD (crime scene DNA, DQA1 locus-specific primers).

Each amplification reaction mixture is heated to 94° C. for 30 seconds.The primers are annealed to the sample DNA by cooling the reactionmixtures to 65° C. for each of the A locus-specific amplificationmixtures and to 55° C. for each of the DQA1 locus-specific amplificationmixtures and maintaining the respective temperatures for one minute. Theprimer extension step is performed by heating each of the amplificationmixtures to 72° C. for one minute. The denaturation, annealing andextension cycle is repeated 30 times for each amplification mixture.

Each amplification mixture is aliquoted to prepare three restrictionendonuclease digestion mixtures per amplification mixture. The A locusreaction mixtures are combined with the endonucleases BsrI, Cfr101 andDraII. The DQA1 reaction mixtures are combined with AluI, CvijI andDdeI.

To produce each digestion mixture, each of three replicate aliquots of10 μl of each amplification mixture is combined with 5 units of therespective enzyme for 60 minutes at 37° C. under conditions recommendedby the manufacturer of each endonuclease.

Following digestion, the three digestion mixtures for each of thesamples (SA, SD, CA and CD) are pooled and electrophoresed on a 6.5%polyacrylamide gel for 45 minutes at 100 volts. Followingelectrophoresis, the gel is stained with ethidium bromide.

The samples contain fragments of the following lengths:

SA: 786, 619, 596, 462, 427, 399, 256, 251, 93, 80

CA: 809, 786, 619, 596, 473, 462, 427, 399, 369, 335, 315, 283, 256,251, 247, 93, 80

SD: 388, 338, 332, 277, 219, 194, 122, 102, 89, 79, 64, 55

CD: 587, 449, 388, 338, 335, 332, 277, 271, 219, 194, 187, 122, 102, 99,89, 88, 79, 65, 64, 55

The analysis demonstrates that the blood from the crime scene and fromthe suspected perpetrator are not from the same individual. The bloodfrom the crime scene and from the suspected perpetrator are,respectively, A3, A9, DQA1 0501, DQA1 0301 and A9, A9, DQA1 0501, DQA10501.

EXAMPLE 2 Paternity Testing

Chorionic villus tissue was obtained by transcervical biopsy from a7-week old conceptus (fetus). Blood samples were obtained byvenepuncture from the mother (M), and from the alleged father (AF). DNAwas extracted from the chorionic villus biopsy, and from the bloodsamples. DNA was extracted from the sample from M by use of nonionicdetergent (Tween 20) and proteinase K. DNA was extracted from the samplefrom F by hypotonic lysis. More specifically, 100 μl of blood wasdiluted to 1.5 ml in PBS and centrifuged to remove buffy coat. Followingtwo hypotonic lysis treatments involving resuspension of buffy coatcells in water, the pellets were washed until redness disappeared.Colorless pellets were resuspended in water and boiled for 20 minutes.Five 10 mm chorionic villus fronds were received. One frond was immersedin 200 μl water. NaOH was added to 0.05 M. The sample was boiled for 20minutes and then neutralized with HCl. No further purification wasperformed for any of the samples.

The extracted DNA was submitted to PCR for amplification of sequencesassociated with the HLA loci, DQA1 and DPB1. The primers used were: (1)as a 5' primer for the DQA1 locus, the primer designated SGD001.DQA1.LNP(DQA 5'IVS1) (corresponding to nt 23-39 of the DQA1 0301 allelesequence) and as the 3' primer for the DQA1 locus, the primer designatedSGD003.DQA1.RNP (DQA 3'IVS2 corresponding to nt 789-806 of the DQA1 0301sequence; (2) as the DPB primers, the primers designated 5'IVS1 nt7604-7624 and 3'IVS2 7910-7929. The amplification reaction mixtureswere: 150 ng of each primer; 25 μ of test DNA; 10 mM Tris HCl, pH 8.3;50 mM KCl; 1.5 mM MgCl₂ ; 0.01% (w/v) gelatin; 200 μm dNTPs; water to100 μl and 2.5 U Taq polymerase.

The amplification was performed by heating the amplification reactionmixture to 94° C. for 10 minutes prior to addition of Taq polymerase.For DQA1, the amplification was performed at 94° C. for 30 seconds, then55° C. for 30 seconds, then 72° C. for 1 minute for 30 cycles, finishingwith 72° C. for 10 minutes. For DPB, the amplification was performed at96° C. for 30 seconds, then 65° C. for 30 seconds, finishing with 65° C.for 10 minutes.

Amplification was shown to be technically satisfactory by test gelelectrophoresis which demonstrated the presence of double stranded DNAof the anticipated size in the amplification reaction mixture. The testgel was 2% agarose in TBE (tris borate EDTA) buffer, loaded with 15 μlof the amplification reaction mixture per lane and electrophoresed at200 v for about 2 hours until the tracker dye migrated between 6 to 7 cminto the 10 cm gel.

The amplified DQA1 and DPB1 sequences were subjected to restrictionendonuclease digestion using DdeI and MboII (8 and 12 units,respectively at 37° C. for 3 hours) for DQA1, and RsaI and FokI (8 and11 units, respectively at 37° C. overnight) for DPB1 in 0.5 to 2.0 μl ofenzyme buffers recommended by the supplier, Pharmacia together with16-18 μl of the amplified product. The digested DNA was fragmentsize-length separated on gel electrophoresis (3% Nusieve). The RFLPpatterns were examined under ultraviolet light after staining the gelwith ethidium bromide.

Fragment pattern analysis is performed by allele assignment of thenon-maternal alleles using expected fragment sizes based on thesequences of known endonuclease restriction sites. The fragment patternanalysis revealed the obligate paternal DQA1 allele to be DQA1 0102 andDPB to be DPw1. The fragment patterns were consistent with AF being thebiological father.

To calculate the probability of true paternity, HLA types were assigned.Maternal and AF DQA1 types were consistent with those predicted from theHLA Class II gene types determined by serological testing usinglymphocytotoxic antisera.

Considering alleles of the two HLA loci as being in linkage equilibrium,the combined probability of non-paternity was given by:

    0.042×0.314-0.013

i.e. the probability of paternity is (1-0.013) or 98.7%.

The relative chance of paternity is thus 74:75, i.e. the chance that theAF is not the biological father is approximately 1 in 75. The parties tothe dispute chose to regard these results as confirming the paternity ofthe fetus by the alleged father.

EXAMPLE 3 Analysis of the HLA DQA1 Locus

The three haplotypes of the HLA DQA1 0102 locus were analyzed asdescribed below. Those haplotypes are DQA1 0102 DR15 Dw2; DQA1 0102 DR16Dw21; and DQA1 0102 DR13 Dw19. The distinction between the haplotypes isparticularly difficult because there is a one basepair differencebetween the 0102 alleles and the 0101 and 0103 alleles, which differenceis not unique in DQA1 allele sequences.

The procedure used for the amplification is the same as that describedin Example 1, except that the amplification used thirty cycles of 94° C.for 30 seconds, 60° C. for 30 seconds, and 72° C. for 60 seconds. Thesequences of the primers were:

SGD 001--5' TTCTGAGCCAGTCCTGAGA 3'; and

SGD 003--5' GATCTGGGGACCTCTTGG 3'.

These primers hybridize to sequences about 500 bp upstream from the 5'end of the second exon and 50 bp downstream from the second exon andproduce amplified DNA sequences in the 700 to 800 bp range.

Following amplification, the amplified DNA sequences wereelectrophoresed on a 4% polyacrylamide gel to determine the PDLP type.In this case, amplified DNA sequences for 0102 comigrate with (are thesame length as) 0101 alleles and subsequent enzyme digestion isnecessary to distinguish them.

The amplified DNA sequences were digested using the restriction enzymeAluI (Bethesda Research Laboratories) which cleaves DNA at the sequenceAGCT. The digestion was performed by mixing 5 units (1 μl) of enzymewith 10 μl of the amplified DNA sequence (between about 0.5 and 1 μg ofDNA) in the enzyme buffer provided by the manufacturer according to themanufacturer's directions to form a digest. The digest was thenincubated for 2 hours at 37° C. for complete enzymatic digestion.

The products of the digestion reaction are mixed with approximately 0.1μg of "ladder" nucleotide sequences (nucleotide control sequencesbeginning at 123 bp in length and increasing in length by 123 bp to afinal size of about 5,000 bp; available commercially from BethesdaResearch Laboratories, Bethesda Md.) and were electrophoresed using a 4%horizontal ultra-thin polyacrylamide gel (E-C Apparatus, ClearwaterFla.). The bands in the gel were visualized (stained) using silver staintechnique [Allen et al, BioTechniques 7:736-744 (1989)].

Three distinctive fragment patterns which correspond to the threehaplotypes were produced using AluI. The patterns (in base pair sizedfragments) were:

1. DR15 DQ6 Dw2: 120, 350, 370, 480

2. DR13 DQ6 Dw19: 120, 330, 350, 480

3. DR16 DQ6 Dw21: 120, 330, 350

The procedure was repeated using a 6.5% vertical polyacrylamide gel andethidium bromide stain and provided the same results. However, thefragment patterns were more readily distinguishable using the ultrathingels and silver stain.

This exemplifies analysis according to the method of this invention.Using the same procedure, 20 of the other 32 DR/DQ haplotypes for DQA1were identified using the same primer pair and two additional enzymes(DdeI and MboII). PDLP groups and fragment patterns for each of the DQA1haplotypes with the three endonucleases are illustrated in Table 6.

    __________________________________________________________________________    AluI                                                                          PDLP                                                                              DR Dw   480                                                                              410                                                                              405                                                                              400                                                                              390                                                                              370                                                                              360                                                                              350                                                                              340                                                                              330                                                                              310                                                                              300                                                                              270                                                                              240                                                                              120                                                                              110                                                                              100               __________________________________________________________________________    0101                                                                              1   1   |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       +  |                                                                       +  |                                                                       |                                                                       +  |            1  20   |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       +  |                                                                       +  |                                                                       |                                                                       +   |           14  9   |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       +  |                                                                       +  |                                                                       |                                                                       +   |       0102                                                                              15  2   +  |                                                                       |                                                                       |                                                                       |                                                                       +  |                                                                       +  |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       +  |                                                                    |               16 21   |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       +  |                                                                       +  |                                                                       |                                                                       |                                                                       |                                                                       +  |                                                                    |               13 19   +  |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       +  |                                                                       +  |                                                                       |                                                                       |                                                                       |                                                                       +  |                                                                    |           0103                                                                              13 18,24                                                                              |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                    |               13 18,25                                                                              |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       +  |                                                                       +  |                                                                       |                                                                       +   |           8   8.3 |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       +  |                                                                       +  |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       +  |                                                                    +                        11 DB2  |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                    |               15 12   |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                    |           0201                                                                              7  DB1  |                                                                       |                                                                       |                                                                       +  |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       +  |                                                                       |                                                                       |                                                                       |                                                                    |               7  17   |                                                                       |                                                                       |                                                                       +  |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       +  |                                                                       |                                                                       |                                                                       |                                                                    |               7  11   |                                                                       |                                                                       |                                                                       |                                                                       +  |                                                                       |                                                                       |                                                                       +  |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                    |           0301                                                                              4   4(7)                                                                              |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                    |               4  13.2(7)                                                                            |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                    |               4   4(8)                                                                              |                                                                       |                                                                       |                                                                       +  |                                                                       |                                                                       |                                                                       +  |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                    |               4  10(8)                                                                              |                                                                       |                                                                       |                                                                       +  |                                                                       |                                                                       |                                                                       +  |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                    |               4  13.1(8)                                                                            |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                    |               4  14(8)                                                                              |                                                                       |                                                                       |                                                                       +  |                                                                       |                                                                       |                                                                       +  |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                    |               4  KT2  |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                    |               4  15   |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                    |               9  23   |                                                                       |                                                                       +  |                                                                       |                                                                       |                                                                       +  |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                    |           0401                                                                              8   8.1 |                                                                       (+)                                                                              |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       +  |                                                                       |                                                                       +  +  +   |           8   8.2 |                                                                       (+)                                                                              |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       +  |                                                                       |                                                                       +  +  +   |           3  RSH  |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                    |           0501                                                                              3   3,24                                                                              |                                                                       (+)                                                                              |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       +  |                                                                       |                                                                       |                                                                       |                                                                       +   |           3   3,25                                                                              |                                                                       (+)                                                                              |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       +  |                                                                       |                                                                       |                                                                       |                                                                       +   |           11  5   |                                                                       (+)                                                                              |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       +  |                                                                       |                                                                       |                                                                       |                                                                       +   |           11  5(9104)                                                                           |                                                                       |                                                                       +  |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       +  |                                                                       |                                                                       |                                                                       |                                                                       +   |           14 16   |                                                                       (+)                                                                              |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       +  |                                                                       |                                                                       |                                                                       |                                                                       +   |           12 DB6  |                                                                       (+)                                                                              |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       +  |                                                                       |                                                                       |                                                                       |                                                                       +   |           16 22   |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                    |           0601                                                                              8   8.3 |                                                                       (+)                                                                              |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       +  |                                                                       |                                                                       |                                                                       |                                                                       +   |       PDLP                                                                              DR Dw   480                                                                              410                                                                              405                                                                              400                                                                              390                                                                              370                                                                              360                                                                              350                                                                              340                                                                              330                                                                              310                                                                              300                                                                              270                                                                              240                                                                              120                                                                              110 100              __________________________________________________________________________

    __________________________________________________________________________    DdeI                                                                          PDLP                                                                              DR Dw   650                                                                              520                                                                              450                                                                              420                                                                              410                                                                              400                                                                              390                                                                              300                                                                              200                                                                              190                                                                              150                                                                              90                                                                              80                                                                              60                                                                              50                         __________________________________________________________________________    0101                                                                              1   1   |                                                                       |                                                                       |                                                                       +  |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       +  |                                                                      + + |                     1  20   |                                                                       |                                                                       |                                                                       +  |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       +  |                                                                      + + |                     14  9   |                                                                       |                                                                       |                                                                       +  |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       +  |                                                                      + + |                 0102                                                                              15  2   |                                                                       +  |                                                                       +  |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       +  |                                                                      + + |                     16 21   |                                                                       |                                                                       |                                                                       +  |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       +  |                                                                      + + |                     13 19   |                                                                       |                                                                       |                                                                       |                                                                       +  |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       +  |                                                                      + + |                 0103                                                                              13 18,24                                                                              |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                      |                                                                      |                                                                      |                     13 18,25                                                                              |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                      |                                                                      |                                                                      |                     8   8.3 |                                                                       |                                                                       +  |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       +  |                                                                      + |                                                                      +                              11 DB2  |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                      |                                                                      |                                                                      |                     15 12   |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                      |                                                                      |                                                                      |                 0201                                                                              7  DB1  |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       +  |                                                                       |                                                                       |                                                                       |                                                                       +  |                                                                      + |                                                                      +                              7  17   |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       +  |                                                                      + |                                                                      +                              7  11   |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       +  |                                                                       |                                                                       |                                                                       |                                                                       +  |                                                                      + |                                                                      +                          0301                                                                              4   4(7)                                                                              |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                      |                                                                      |                                                                      |                     4  13.2(7)                                                                            |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                      |                                                                      |                                                                      |                     4   4(8)                                                                              +  |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       + |                                                                      |                                                                      +                              4  10(8)                                                                              +  |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       + |                                                                      |                                                                      +                              4  13.1(8)                                                                            |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                      |                                                                      |                                                                      |                     4  14(8)                                                                              +  |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       + |                                                                      |                                                                      +                              4  KT2  |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                      |                                                                      |                                                                      |                     4  15   |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                      |                                                                      |                                                                      |                     9  23   +  |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       + |                                                                      |                                                                      +                          0401                                                                              8   8.1 |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       +  +  |                                                                       |                                                                       + + |                                                                      +                              8   8.2 |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       +  +  |                                                                       |                                                                       + + |                                                                      +                              3  RSH  |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                      |                                                                      |                                                                      |                 0501                                                                              3   3,24                                                                              |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       +  |                                                                       |                                                                       +  |                                                                       |                                                                       |                                                                      + |                                                                      +                              3   3,25                                                                              |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       +  |                                                                       |                                                                       +  |                                                                       |                                                                       |                                                                      + |                                                                      +                              11  5   |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       +  |                                                                       |                                                                       +  |                                                                       |                                                                       |                                                                      + |                                                                      +                              11  5(9104)                                                                           |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       +  |                                                                       |                                                                       |                                                                       +  |                                                                       |                                                                      + |                                                                      +                              14 16   |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       +  |                                                                       +  |                                                                       |                                                                       |                                                                      + |                                                                      +                              12 DB6  |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       +  |                                                                       |                                                                       +  |                                                                       |                                                                       |                                                                      + |                                                                      +                              16 22   |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                      |                                                                      |                                                                      |                 0601                                                                              8   8.3 |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       |                                                                       +  +  |                                                                       |                                                                       + + |                                                                      +                          PDLP                                                                              DR Dw   650                                                                              520                                                                              450                                                                              420                                                                              410                                                                              400                                                                              390                                                                              300                                                                              200                                                                              190                                                                              150                                                                              90                                                                              80                                                                              60                                                                              50                         __________________________________________________________________________

      MboII PDLP DR Dw 390 385 380 370 365 360 350 340 335 330 305 300 250     190 180 170 140 130       0101 1  1 | | | | |     | + | | | | + |     | | | | |  1 20 |     | | | | | + |     | | | + | | |     | | |  14  9 | | |     | | | + | | | +     | | | | | |     | 0102 15  2 | | | + |     | | | | | | +     | | | | | |  16 21     | | | + | | |     | | | | + | |     | | | |  13 19 | |     | + | | | | |     | | + | | | |     | | 0103 13 18,24 | | |     | | | | | |     | | | | | |     | | |  13 18,25 | |     | | | + | | |     | | | | + | | +     |  8      8.3 | | | | | + |     | | | | | | +     | | + |  11 DB2 | |     | | | | | |     | | | | | |     | | | |  15 12 | |     | | | | | |     | | | | | |     | | | | 0201 7 DB1 |     | | | | | + |     | | | | | |     | + | +  7 17 | | |     | | | + | | |     | | | | | + | +  7     11 | | | | | | +     | |  | | | |     | | + | + 0301 4  4(7) | |     | | | | | |     | | | | | |     | | | |  4 13.2(7) |     | | | | | |     | | | | | |     | | | | |  4      4(8) | | | | | |     | | | | | | +     | + + | |  4 10(8) | |     | | | | | |     | | | | + | + + |     |  4 13.1(8) | | | |     | | | | | |     | | | | | |     | |  4 14(8) | | | .vertline     . | | | | | |     | | + | + + | |  4 KT2     | | | | | |     | | | | | |     | | | | | |  4 15     | | | | | |     | | | | | |     | | | | | |  9 23     | | | | | |     | | | | | | +     | + + | | 0401 8  8.1 | |     | | | + | | | +     | | | | | |     | |  8      8.2 | | | | + | |     | + | | | | |     | | | |  3 RSH | |     | | | | | |     | | | | | |     | | | | 0501 3  3,24 + | +     | | | | + | |     | | | | | |     | |  3  3,25 + | + | |     | | + | | | |     | | | | | |  11  5     + | + | | | | + |     | | | |  | |     | | |  11  5(9104) | |     | | | | | + |     | | | | | |     | + +  14 16 | + | | |     | | | | | + |     | | | | | |  12     DB6 + | + | | | | +     | | | | | |     | | | |  16 22 | |     | | | | | |     | | | | | |     | | | | 0601 8  8.3 |     | | | | | |     | | | + | | |     | | | | PDLP DR Dw 390 385 380 370     365 360 350 340 335 330 305 300 250 190 180 170 140 130

This example illustrates the ability of the method of this invention todistinguish the alleles and haplotypes of a genetic locus. Specifically,the example shows that PDLP analysis stratifies five of the eightalleles. These three restriction endonuclease digests distinguish eachof the eight alleles and many of the 35 known haplotypes of the locus.The use of additional endonuclease digests for this amplified DNAsequence can be expected to distinguish all of the known haplotypes andto potentially identify other previously unrecognized haplotypes.Alternatively, use of the same or other endonuclease digests for anotheramplified DNA sequence in this locus can be expected to distinguish thehaplotypes.

In addition, analysis of amplified DNA sequences at the DRA locus in thetelomeric direction and DQB in the centromeric direction, preferablytogether with analysis of a central locus, can readily distinguish allof the haplotypes for the region.

The same methods are readily applied to other loci.

EXAMPLE 4

Analysis of the HLA DQA1 Locus

The DNA of an individual is analyzed to determine which of the threehaplotypes of the HLA DQA1 0102 locus are present. Genomic DNA isamplified as described in Example 3. Each of the amplified DNA sequencesis sequenced to identify the haplotypes of the individual. Theindividual is shown to have the haplotypes DR15 DQ6 Dw2; DR13 DQ6 Dw19.

The procedure is repeated as described in Example 3 through theproduction of the AluI digest. Each of the digest fragments issequenced. The individual is shown to have the haplotypes DR15 DQ6 Dw2;DR13 DQ6 Dw19.

EXAMPLE 5 DQA1 Allele-Specific Amplification

Primers were synthesized that specifically bind the 0102 and 0301alleles of the DQA1 locus. The 5' primer was the SGD 001 primer used inExample 3. The sequences of the 3' primers are listed below.

0102 5' TTGCTGAACTCAGGCCACC 3'

0301 5' TGCGGAACAGAGGCAACTG 3'

The amplification was performed as described in Example 3 using 30cycles of a standard (94° C., 60° C., 72° C.) PCR reaction. The templateDNAs for each of the 0101, 0301 and 0501 alleles were amplifiedseparately. As determined by gel electrophoresis, the0102-allele-specific primer amplified only template 0102 DNA and the0301-allele-specific primer amplified only template 0301 DNA. Thus, eachof the primers was allele-specific.

EXAMPLE 6 Detection of Cystic Fibrosis

The procedure used for the amplification described in Example 3 isrepeated. The sequences of the primers are illustrated below. The firsttwo primers are upstream primers, and the third is a downstream primer.The primers amplify a DNA sequence that encompasses all of interveningsequence 1

5' CAG AGG TCG CCT CTG GA 3';

5' AAG GCC AGC GTT GTC TCC A 3'; and

3' CCT CAA AAT TGG TCT GGT 5'.

These primers hybridize to the complement of sequences located from nt136-152 and nt 154-172, and to nt 187-207. [The nucleotide numbers arefound in Riordan et al, Science 245:1066-1072 (1989).]

Following amplification, the amplified DNA sequences are electrophoresedon a 4% polyacrylamide gel to determine the PDLP type. The amplified DNAsequences are separately digested using each of the restriction enzymesAluI, MnlI and RsaI (Bethesda Research Laboratories). The digestion isperformed as described in Example 3. The products of the digestionreaction are electrophoresed and visualized using a 4% horizontalultra-thin polyacrylamide gel and silver stain as described in Example3.

Distinctive fragment patterns which correspond to disease-associated andnormal haplotypes are produced.

EXAMPLE 7 Analysis of Bovine Leukocyte Antigen Class I

Bovine Leukocyte Antigen (BoLA) Class I alleles and haplotypes areanalyzed in the same manner as described in Example 3. The primers arelisted below.

    __________________________________________________________________________    Bovine Primers (Class I HLA homolog)                                                                         T.sub.m                                        __________________________________________________________________________    5' primer:                                                                            5'                                                                              TCC TGG TCC TGA CCG AGA 3'                                                                         (62°)                                   3' primer:                                                                            1)                                                                              3' A TGT GCC TTT GGA GGG TCT 5'                                                                    (62°)                                             (for .sup.˜ 600 bp product)                                           2)                                                                              3' GCC AAC AT GAT CCG CAT 5'                                                                       (62°)                                             (for ˜900 bp product)                                         __________________________________________________________________________

For the approximately 900 bp sequence PDLP analysis is sufficient todistinguish alleles 1 and 3 (893 and 911 bp, respectively). Digests areprepared as described in Example 3 using AluI and DdeI. The followingpatterns are produced for the 900 bp sequence.

Allele 1, AluI digest: 712, 181

Allele 3, AluI digest: 430, 300, 181

Allele 1, DdeI digest: 445, 201, 182, 28

Allele 3, DdeI digest: 406, 185, 182, 28, 16

The 600 bp sequence also produces distinguishable fragment patterns forthose alleles. However, those patterns are not as dramatically differentas the patterns produced by the 600 bp sequence digests.

EXAMPLE 8 Preparation of Primers

Each of the following primers is synthesized using an Applied Biosystemsmodel 308A DNA synthesizer.

    __________________________________________________________________________    HLA locus primers                                                             __________________________________________________________________________             A locus-specific primers                                                      SGD009.AIVS3.R2NP CATGTGGCCATCTTGAGAATGGA                                     SGD006.AIVS3.R1NP GCCCGGGAGATCTACAGGCGATCA                                    A2.1 CGCCTCCCTGATCGCCTGTAG                                                    A2.2 CCAGAGAGTGACTCTGAGG                                                      A2.3 CACAATTAAGGGAT                                                           B locus-specific primers                                                      SGD007.BIVS3.R1NP TCCCCGGCGACCTATAGGAGATGG                                    SGD010.BIVS3.R2NP CTAGGACCACCCATGTGACCAGC                                     B2.1  ATCTCCTCAGACGCCGAGATGCGTCAC                                             B2.2  CTCCTGCTGCTCTGGGGGGCAG                                                  B2.3  ACTTTACCTCCACTCAGATCAGGAG                                               B2.4  CGTCCAGGCTGGTGTCTGGGTTCTGTGCCCCT                                        B2.5  CTGGTCACATGGGTGGTCCTAGG                                                 B2.6  CGCCTGAATTTTCTGACTCTTCCCAT                                              C locus-specific primers                                                      SGD008.CIVS3.R1NP ATCCCGGGAGATCTACAGGAGATG                                    SGD011.CIVS3.R2NP AACAGCGCCCATGTGACCATCCT                                     C2.1  CTGGGGAGGCGCCGCGTTGAGGATTCT                                             C2.2  CGTCTCCGCAGTCCCGGTTCTAAAGTTCCCAGT                                       C2.3  ATCCTCGTGCTCTCGGGA                                                      C2.4  TGTGGTCAGGCTGCTGAC                                                      C2.5  AAGGTTTGATTCCAGCTT                                                      C2.6  CCCCTTCCCCACCCCAGGTGTTCCTGTCCATTCTTCAGGA                                C2.7  CACATGGGCGCTGTTGGAGTGTCG                                                Class I loci-specific primers                                                 SGD005.IIVS1.LNP GTGAGTGCGGGGTCGGGAGGGA                                       1.1 CACCCACCGGGACTCAGA                                                        1.2 TGGCCCTGACCCAGACCTGGGC                                                    1.3 GAGGGTCGGGCGGGTCTCAGC                                                     1.4 CTCTCAGGCCTTGTTC                                                          1.5 CAGAAGTCGCTGTTCC                                                          DOA1 locus-specific primers                                                   SGD001.DQA1.LNP TTCTGAGCCAGTCCTGAGA                                           DQA3 E1a TTGCCCTGACCACCGTGATG                                                 DQA3 E1b CTTCCTGCTTGTCATCTTCA                                                 DQA3 E1c CCATGAATTTGATGGAGA                                                   DQA3 E1d ACCGCTGCTACCAATGGTA                                                  SGD003.DQA1.RNP CCAAGAGGTCCCCAGATC                                            DRA locus-specific primers                                                    DRA E1 TCATCATAGCTGTGCTGATG                                                   DRA 5'E2 AGAACATGTGATCATCCAGGC                                                DRA 3'E2 CCAACTATACTCCGATCACCAAT                                              DRB locus-specific primers                                                    DRB E1 TGACAGTGACACTGATGGTGCTG                                                DRB 5'E2 GGGGACACCCGACCACGTTTC                                                DRB 3'E2 TGCAGACACAACTACGGGGTTG                                               DQB1 locus-specific primers                                                   DQB E1 TGGCTGAGGGCAGAGACTCTCCC                                                DQB 5'E2 TGCTACTTCACCAACGGGAC                                                 DQB 3'E2 GGTGTGCACACACAACTAC                                                  DQB 5'IVS1a AGGTATTTTACCCAGGGACCAAGAGAT                                       DQB 5'IVS1b ATGTAAAATCAGCCCGACTGCCTCTTC                                       DQB 3'IVS2 GCCTCGTGCCTTATGCGTTTGCCTCCT                                        DPB1 locus-specific primers                                                   DPB E1 TGAGGTTAATAAACTGGAGAA                                                  DPB 5'IVS1 GAGAGTGGCGCCTCCGCTCAT                                              DPB 3'IVS2 GAGTGAGGGCTTTGGGCCGG                                      __________________________________________________________________________

What is claimed is:
 1. A method for detection of at least one codingregion allele of an HLA locus comprising amplifying genomic DNA with aprimer pair that spans a non-coding region sequence selected from thegroup consisting of untranslated sequences between exons, 5' and 3'untranslated regions associated with a genetic locus, and spacingsequences between genetic loci, said primer pair defining a DNAsequence, said DNA sequence being in genetic linkage with said HLA locusand containing a sufficient number of said non-coding region sequencenucleotides to produce an amplified DNA sequence characteristic of saidallele.
 2. The method of claim 1 wherein said amplified DNA sequenceincludes at least about 300 nucleotides corresponding to intronsequences.
 3. The method of claim 1 wherein said intron sequence isadjacent to an exon encoding said allele.
 4. The method of claim 1wherein said amplified DNA sequence is characteristic of at least onenonadjacent allele.
 5. The method of claim 1 wherein said amplified DNAsequence is characteristic of at least one adjacent allele and at leastone nonadjacent allele.
 6. The method of claim 5 wherein said amplifiedDNA sequence includes at least about 1,000 nucleotides corresponding tonon-coding region sequences.
 7. A method for detection of at least oneallele of an HLA locus comprising:A. amplifying genomic DNA with aprimer pair that spans a non-coding region sequence selected from thegroup consisting of untranslated sequences between exons, 5' and 3'untranslated regions associated with a genetic locus, and spacingsequences between genetic loci, said primer pair defining a DNAsequence, said DNA sequence being in genetic linkage with said alleleand containing a sufficient number of said non-coding region sequencenucleotides to produce an amplified DNA sequence characteristic of saidallele; and B. analyzing said amplified DNA sequence to detect thepresence of a genetic variation in said amplified sequence.
 8. Themethod of claim 7 wherein said variation in said amplified DNA sequenceis a variation in the length of the primer-defined amplified DNAsequence.
 9. The method of claim 7 wherein said variation in saidamplified DNA sequence is a change in the presence of at least onerestriction site in the primer-defined amplified DNA sequence.
 10. Themethod of claim 7 wherein said variation in said amplified DNA sequenceis a change in the location of at least one restriction site in theprimer-defined amplified DNA sequence.
 11. The method of claim 7 whereinsaid variation in said amplified DNA sequence is a substitution of atleast one nucleotide in the primer-defined amplified DNA sequence.